Note: Descriptions are shown in the official language in which they were submitted.
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8-ETHYL-6-(ARYL)PYRIDO[2,3-D]PYRIMIDIN-7(8H)-ONES FOR THE
TREATMENT OF CNS DISORDERS
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No.
61/250,262, filed October 9, 2009, and U.S. Provisional Application No.
61/353,054, filed
June 9, 2010, which are both incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Central Nervous System (CNS) disorders are characterized by a variety
of
debilitating affective and cognitive impairments. For example, a clinical sign
of individuals
with Alzheimer's disease is progressive cognition deterioration. Worldwide,
approximately
24 million people have dementia, 60% of these cases are due to Alzheimer's.
[0003] Other CNS disorders include, e.g., mood disorders, age-related
cognitive decline,
and neurological disorders (e.g., epilepsy, schizophrenia, Fragile X mental
retardation
syndrome and Huntington's disease). The effects of CNS disorders are
devastating to the
quality of life of those afflicted as well as that of their families.
Moreover, CNS disorders
impose an enormous health care burden on society. A number of CNS disorders,
as well as
other conditions that affect cognitive function, have been associated with
alterations in the
morphology and/or density of dendritic spines, membranous protrusions from
dendritic
shafts of neurons that serve as highly specialized structures for the
formation, maintenance,
and function of synapses.
SUMMARY OF THE INVENTION
[0004] Described herein are compounds, compositions and methods for treating
an
individual suffering from a CNS disorder, such as by way of example only
schizophrenia,
Fragile X Syndrome (FXS), clinical depression, age-related cognitive decline,
Mild
Cognitive Impairment, Huntington's disease, Parkinson's disease,
neurofibromatosis,
Alzheimer's disease, epilepsy, autism spectrum disorders, mental retardation,
Down's
syndrome or the like, by administering to an individual a pharmaceutical
composition
comprising a therapeutically effective amount of an inhibitor of a p21-
activated kinase
(PAK), e.g., an inhibitor of PAK1, PAK2, PAK3 or PAK4, as described herein.
PAK
activation is shown to play a key role in spine morphogenesis. In some
instances,
attenuation of PAK activity reduces, prevents or reverses defects in spine
morphogenesis. In
some embodiments, inhibitors of one or more of Group I PAKs (PAK1, PAK2 and/or
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PAK3) and/or Group II PAKs (PAK4, PAK5 and/or PAK6) are administered to rescue
defects in spine morphogenesis in individuals suffering from a condition in
which dendritic
spine morphology, density, and/or function are aberrant, including but not
limited to
abnormal spine density, spine size, spine shape, spine plasticity, spine
motility or spine
plasticity leading to improvements in synaptic function, cognition and/or
behavior.
[0005] In one aspect is a compound having the structure of Formula I or
pharmaceutically acceptable salt or N-oxide thereof:
R7
(R5)r B
N N N 0
H I
Q
Formula I;
wherein:
(R4)s
T
R7 is z R3
wherein ring T is an aryl, or a heteroaryl ring;
R3 is a substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted
heteroaryl attached to ring T via a carbon atom of R3, or a substituted or
unsubstituted heterocycloalkyl attached to ring T via a carbon atom of R3;
Q is a substituted or unsubstituted alkyl, a substituted or unsubstituted
heteroalkyl, a
substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted
cycloalkyl, a substituted or unsubstituted cycloalkylalkyl, a substituted or
unsubstituted heterocycloalkylalkyl, a substituted or unsubstituted aryl, a
substituted or unsubstituted arylalkyl, a substituted or unsubstituted
heteroaryl,
or a substituted or unsubstituted heteroarylalkyl;
each R4 is independently halogen, -CN, -NO2, -OH, -OCF3, -OCH2F, -OCF2H, -CF3,
-SR8, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -OC(=O)R9, -CO2R10,
-N(R10)2, -C(=O)N(R10)2, -NR10C(=O)R10, -N R10C(=O)OR10,
-NR10C(=O)N(R10)2, a substituted or unsubstituted alkyl, a substituted or
unsubstituted alkoxy, a substituted or unsubstituted heteroalkyl, a
substituted or
unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl;
R8 is H or R9;
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R9 is a substituted or unsubstituted alkyl, a substituted or unsubstituted
cycloalkyl, a
substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted
aryl,
or a substituted or unsubstituted heteroaryl;
each R10 is independently H, a substituted or unsubstituted alkyl, a
substituted or
unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a
substituted or unsubstituted aryl, or a substituted or unsubstituted
heteroaryl; or
two R10, together with the atoms to which they are attached form a
heterocycle;
ring B is aryl or heteroaryl;
each R5 is independently halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -S(=O)2R9,
NR10S =0 2R9 S =0 2N R10 C =0 R8 OC =0 R9 C02R10 N R1o
C(0)N(R10)2, NR10C(=0)R10 NR10C(=O)OR10 NR10C(=O)N(R10)2, -OR10, a
substituted or unsubstituted alkyl, a substituted or unsubstituted alkoxy, a
substituted or unsubstituted heteroalkyl, a substituted or unsubstituted
cycloalkyl, or a substituted or unsubstituted heterocycloalkyl;
r is 0 to 8; and
s is 0 to 4.
[0006] In one embodiment is a compound of Formula I wherein ring T is an aryl
ring.
In another embodiment is a compound of Formula I wherein ring T is a
heteroaryl ring. In
yet another embodiment is a compound of Formula I, wherein ring T is selected
from
pyrrole, furan, thiophene, pyrazole, imidazole, isoxazole, oxazole,
isothiazole, thiazole,
1,2,3-triazole, 1,3,4-triazole, 1-oxa-2,3-diazole, 1-oxa-2,4-diazole, 1-oxa-
2,5-diazole, 1-oxa-
3,4-diazole, 1-thia-2,3-diazole, 1-thia-2,4-diazole, 1-thia-2,5-diazole, 1-
thia-3,4-diazole,
tetrazole, pyridine, pyridazine, pyrimidine, and pyrazine. In yet a further
embodiment is a
compound of Formula I, wherein R3 is a C-linked heterocycloalkyl. In another
embodiment
is a compound of Formula I, wherein R3 is a substituted or unsubstituted C-
linked
heteroaryl. In another embodiment, R3 is a substituted or unsubstituted
cycloalkyl. In a
further embodiment, cycloalkyl is selected from cyclopropyl, cyclobutyl,
cyclopentyl, or
cyclohexyl.
[0007] In yet another embodiment is a compound having the structure of Formula
II:
3
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R3
T
~~ N (R4 )s
(R5), \ ~I
`~ N N i O
H
Q
Formula II.
[0008] In a further embodiment is a compound having the structure of Formula
III:
(R4)s1 (R4)s1
T
N R3
(R5)r
N N N O
H
Formula III;
wherein s 1 is 0 to 3.
[0009] In yet a further embodiment is a compound of Formula III having the
structure
of Formula IV:
R3
-(R4)S1
N
(R5),
N N N O
H
Formula IV;
wherein s 1 is 0 to 4.
[0010] In another embodiment is a compound of Formula III having the structure
of
Formula V:
R3
IT(R4)1
N \ \
(R5),
N N N O
H
Formula V;
wherein s 1 is 0 to 4.
[0011] In another embodiment is a compound of Formula III having the structure
of
Formula Va:
4
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R3
(R4),
N
(R5), N N N O
H
Formula Va;
wherein s 1 is 0 to 4.
[0012] In another embodiment is a compound of Formula III having the structure
of
Formula Vb:
R4 R3
N
(R5), N N N O
H
Formula Vb.
[0013] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or Vb
wherein R3 is selected from pyrrole, furan, thiophene, pyrazole, imidazole,
isoxazole,
oxazole, isothiazole, thiazole, 1,2,3-triazole, 1,3,4-triazole, 1-oxa-2,3-
diazole, 1-oxa-2,4-
diazole, 1-oxa-2,5-diazole, 1-oxa-3,4-diazole, 1-thia-2,3-diazole, 1-thia-2,4-
diazole, 1-thia-
2,5-diazole, 1-thia-3,4-diazole, tetrazole, pyridine, pyridazine, pyrimidine,
and pyrazine.
[0014] In a further embodiment is a compound of Formula I, II, III, IV, V, Va,
or Vb,
wherein (R5)r-@ is
5
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\ \ 0 f `T N f `T N,
(R5)r I / (R5)r i / / (R5)r / (R5)r I / / (R5)r i / N
f `T N r `T N f T O~ rr Ss (N Y N
(R5)r li 1N (R5)r LI /1 /> (R5)r N (R5)r (R
N \~ `N ,
N__'N 5 rN (N (NN 5 rNYN
(R )r I / (R )r N / (R5), (R5), (R )r N
N N
(NN, 5 N mi l ' N' 5 N, NYS
(R5)r N (R )r / N (R )r N (R5)r II I N (R5)r
N
NS fN~ \ \ \ i \ \
(R5)r N (R5)r / / (R5)r N (R5)r or (R5)r
N N
[0015] In another embodiment is a compound of Formula I, II, III, IV, V, Va,
or Vb,
where R5 is halogen, -CN, -OH, a substituted or unsubstituted alkyl, -OR10, -
NR10S(=O)2R9,
-S(=O)2N(R10)2, -N(R10)2, -C(=O)N(R10)2, -NR10C(=O)R10, -NR10C(=O)OR10,
5 -NR10C(=O)N(R10)2, or a substituted or unsubstituted heterocycloalkyl.
[0016] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein at least one R5 is -NR10S(=O)2R9, -S(=O)2N(R10)2, -N(R10)2, -
C(=O)N(R10)2, -
NR10C(=O)R10, -NR10C(=O)OR10, -NR10C(=O)N(R10)2, or a substituted or
unsubstituted
heterocycloalkyl.
[0017] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein at least one R5 is -N(R10)2, or a substituted or unsubstituted
heterocycloalkyl. In a
further embodiment is a compound of Formula I, II, III, IV, V, Va, or Vb
wherein at least
one of R5 is a substituted or unsubstituted piperazine, a substituted or
unsubstituted
piperidine, a substituted or unsubstituted pyrrolidine, or a substituted or
unsubstituted
morpholine. In one embodiment is a compound of Formula I, II, III, IV, V, Va,
or Vb,
wherein at least one R5 is -OR10. In another embodiment is a compound of
Formula I, II,
III, IV, V, Va, or Vb, wherein R4 is independently halogen, -CN, -OH, -OCF3, -
OCF3, -
OCF2H, -CF3, -SR8, a substituted or unsubstituted alkyl, or a substituted or
unsubstituted
alkoxy.
[0018] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein s is zero.
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[0019] In a further embodiment is a compound of Formula I, II, III, IV, V, Va,
or Vb,
wherein Q is a substituted or unsubstituted alkyl, or a substituted or
unsubstituted
heteroalkyl. In another embodiment is a compound of Formula I, II, III, IV, V,
Va, or Vb,
wherein Q is a substituted or unsubstituted cycloalkyl, or a substituted or
unsubstituted
heterocycloalkyl. In a further embodiment is a compound of Formula I, II, III,
IV, V, Va, or
Vb, wherein Q is a substituted or unsubstituted cycloalkylalkyl, or a
substituted or
unsubstituted heterocycloalkylalkyl. In one embodiment is a compound of
Formula I, II,
III, IV, V, Va, or Vb, wherein Q is a substituted or unsubstituted aryl, or a
substituted or
unsubstituted heteroaryl.
[0020] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein Q is a substituted or unsubstituted arylalkyl, or a substituted or
unsubstituted
heteroarylalkyl.
[0021] Provided herein are pharmaceutical compositions comprising a
therapeutically
effective amount of a compound of Formula I, II, III, IV, V, Va, or Vb, or a
pharmaceutically acceptable salt or N-oxide thereof, and a pharmaceutically
acceptable
carrier, wherein the compound of Formula I-XV is as described herein.
[0022] Provided herein, in some embodiments, are methods for treating CNS
disorders
comprising administering to an individual in need thereof a therapeutically
effective amount
of a compound of Formula I-XV wherein compounds of Formula I-XV are as
described
herein.
[0023] Also provided herein, in some embodiments, are methods for treating
neuropsychiatric conditions comprising administering to an individual in need
thereof a
therapeutically effective amount of a compound of Formula I-XV wherein
compounds of
Formula I-XV are as described herein.
[0024] Also provided herein, in some embodiments, are methods for treating
neurodegenerative disorder comprising administering to an individual in need
thereof a
therapeutically effective amount of a compound of Formula I-XV wherein
compounds of
Formula I-XV are as described herein.
[0025] Also provided herein, in some embodiments, are methods for treating
neurodevelopmental disorder comprising administering to an individual in need
thereof a
therapeutically effective amount of a compound of Formula I-XV wherein
compounds of
Formula I-XV are as described herein.
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[0026] Provided herein, in some embodiments, are methods of modulating a p21-
activated kinase comprising contacting a p21-activated kinase with a compound
of Formula
I-XV.
[0027] In some embodiments of any of the above methods, compounds of any of
Formula I-XV are inhibitors of p21-activated kinase. In some embodiments,
compounds of
any of Formula I-XV inhibit one or more of PAKI, PAK2, PAK3, PAK4, PAK5 or
PAK6.
In some embodiments of any of the above methods compounds of any of Formula I-
XV
inhibit one or more of PAKI, PAK2 or PAK3. In some embodiments of any of the
above
methods, compounds of any of Formula I-XV inhibit PAKI and PAK3. In some
embodiments of any of the above methods, compounds of any of Formula I-XV
inhibit
PAKI and PAK2. In some embodiments of any of the above methods, compounds of
any of
Formula I-XV inhibit PAKI, PAK2 and PAK3. In some embodiments of any of the
above
methods, compounds of any of Formula I-XV inhibit PAKI and PAK4. In some
embodiments of any of the above methods, compounds of any of Formula I-XV
inhibit
PAKI, PAK2, PAK3 and PAK4.
[0028] In some embodiments of any of the above methods, compounds of any of
Formula I-XV inhibit PAKI. In some embodiments of any of the above methods,
compounds of any of Formula I-XV inhibit PAK2. In some embodiments of any of
the
above methods, compounds of any of Formula I-XV inhibit PAK3. In some
embodiments
of any of the above methods, compounds of any of Formula I-XV inhibit PAK4.
[0029] In some embodiments of any of the above methods, a therapeutically
effective
amount of compounds of any of Formula I-XV causes substantially complete
inhibition of
one or more Group I p21-activated kinases.
[0030] In some embodiments of any of the above methods, a therapeutically
effective
amount of compounds of any of Formula I-XV causes partial inhibition of one or
more
Group I p21-activated kinases.
[0031] In one embodiment the CNS disorder is a neurodegenerative disorder, a
neurodevelopmental disorder or a neuropsychiatric disorder.
[0032] In some embodiments of any of the above methods, the neuropsychiatric
disorder is a psychotic disorder, a mood disorder or cognitive impairment.
[0033] In some embodiments of any of the above methods, the CNS disorder is
Schizophrenia, Psychotic disorder, schizoaffective disorder, schizophreniform,
Alzheimer's
disease, Age-related cognitive decline, Mild cognitive impairment, cognitive
decline
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associated with menopause, Parkinson's Disease, Huntington's Disease,
Substance abuse
and substance dependence, Fragile X, Rett's syndrome, Angelman Syndrome,
Asperger's
Syndrome, Autism, Autism Spectrum Disorders, Neurofibromatosis I,
Neurofibromatosis II,
Tuberous sclerosis, Clinical Depression, Bipolar Disorder, Mania, Epilepsy,
Mental
retardation, Down's syndrome, Niemann-Pick disease, Spongiform encephalitis,
Lafora
disease, Maple syrup urine disease, maternal phenylketonuria, atypical
phenylketonuria,
Generalized Anxiety Disorder, Lowe Syndrome, Turner Syndrome, Obsessive-
compulsive
disorder, Panic disorder, Phobias, Posttraumatic Stress Disorder, Anorexia
Nervosa, and
Bulimia Nervosa.
[0034] In some embodiments of any of the above methods, compounds of any of
Formula I-XV modulate dendritic spine morphology or synaptic function. In some
embodiments of any of the above methods, compounds of any of Formula I-XV
modulate
dendritic spine density. In some embodiments of any of the above methods,
compounds of
any of Formula I-XV modulate dendritic spine length. In some embodiments of
any of the
above methods, compounds of any of Formula I-XV modulate dendritic spine neck
diameter. In some embodiments of any of the above methods, compounds of any of
Formula I-XV modulate dendritic spine head volume. In some embodiments of any
of the
above methods, compounds of any of Formula I-XV modulate dendritic spine head
diameter. In some embodiments of any of the above methods, compounds of any of
Formula I-XV modulate the ratio of the number of mature spines to the number
of immature
spines. In some embodiments of any of the above methods, compounds of any of
Formula I-
XV modulate the ratio of the spine head diameter to spine length. In some
embodiments of
any of the above methods, compounds of any of Formula I-XV modulate synaptic
function.
[0035] In some embodiments of any of the above methods, compounds of any of
Formula I-XV normalize or partially normalize aberrant baseline synaptic
transmission
associated with a CNS disorder. In some embodiments of any of the above
methods,
compounds of any of Formula I-XV normalize or partially normalize aberrant
synaptic
plasticity associated with a CNS disorder. In some embodiments of any of the
above
methods, compounds of any of Formula I-XV normalize or partially normalize
aberrant
long term depression (LTD) associated with a CNS disorder. In some embodiments
of any
of the above methods, compounds of any of Formula I-XV normalize or partially
normalize
aberrant long term potentiation (LTP) associated with a CNS disorder.
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[0036] In some embodiments of any of the above methods, compounds of any of
Formula I-XV normalize or partially normalize aberrant sensorimotor gating
associated
with a CNS disorder such as a neuropsychiatric disorder. In some embodiments
of any of
the above methods, compounds of any of Formula I-XV reduce or reverse negative
symptoms associated with a CNS disorder. In some of such embodiments, the
negative
symptoms associated with a CNS disorder are asociality, blunted affect,
avolition, alogia,
anhedonia or dysphoric mood. In some embodiments of any of the above methods,
compounds of any of Formula I-XV reduce or reverse positive symptoms
associated with a
CNS disorder. In some of such embodiments, the positive symptoms associated
with a CNS
disorder are auditory, visual or tactile hallucinations.
[0037] In some embodiments of any of the above methods, compounds of any of
Formula I-XV reduce or reverse cognitive symptoms associated with a CNS
disorder. In
some of such embodiments, the cognitive symptoms associated with a CNS
disorder are
impairment in executive function, comprehension, inference, decision-making,
planning,
learning or memory.
[0038] In some embodiments of any of the above methods compounds of any of
Formula I-XV halt or delay progression of cognitive impairment associated with
a CNS
disorder. In some of such embodiments, the cognitive impairment is mild
cognitive
impairment. In some embodiments, the cognitive impairment is associated with
Alzheimer's
disease.
[0039] In some embodiments of any of the above methods, compounds of any of
Formula I-XV reduce or reverse behavioral symptoms associated with a CNS
disorder. In
some of such embodiments, behavioral symptoms include, for example, repetitive
behavior
(stereotypy), hypersensitivity, hyperactivity, impaired social interaction,
autism or the like.
[0040] In some embodiments of any of the above methods, the method further
comprises administration of a second therapeutic agent that alleviates one or
more
symptoms associated with a CNS disorder.
[0041] In some embodiments, the second therapeutic agent is an antipsychotic
agent, a
cognition enhancer, a Group I mGluR antagonist, a mGluR5 antagonist, a mGluR5
potentiator, a nootropic agent, an alpha7 nicotinic receptor agonist, an
allosteric alpha7
nicotinic receptor potentiator, a nootropic agent, a trophic agent, an
antioxidant, a
neuroprotectant, a beta secretase inhibitor, a gamma secretase inhibitor or an
Abeta
antibody.
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[0042] In some embodiments, administration of a therapeutically effect amount
of
compounds of any of Formula I-XV to an individual in need thereof improves one
or more
of MATRICS cognition scores, Wisconsin Card Sort test scores, Mini-Mental
State Exam
(MMSE) scores, Alzheimer Disease Assessment Scale-Cognitive (ADAS-cog) scale
scores,
ADAS-Behav scores, or Hopkins Verbal Learning Test Revised scores for the
individual.
[0043] Provided herein are methods for reversing cortical hypofrontality
associated with
a CNS disorder comprising administering to an individual in need thereof a
therapeutically
effective amount of a compound of any of Formula I-XV. Provided herein are
methods for
reducing, stabilizing, or reversing neuronal withering and/or loss of synaptic
function
associated a CNS disorder comprising administering to an individual in need
thereof a
therapeutically effective amount of a compound of any of Formula I-XV.
Provided herein
are methods for reducing, stabilizing or reversing atrophy or degeneration of
nervous tissue
in the brain associated with a CNS disorder comprising administering to an
individual in
need thereof a therapeutically effective amount of a compound of any of
Formula I-XV.
[0044] Provided herein are methods of inhibiting the activity of one or more
p21-
activated kinases comprising contacting the one or more p21-activated kinases
with a
compound of any of Formula I-XV. In some embodiments, the one or more p21-
activated
kinase is contacted with a compound of any of Formula I-XV in vitro. In some
embodiments, the one or more p21-activated kinase is contacted with a compound
of any of
Formula I-XV in vivo.
[0045] Provided herein is the use of compounds of any of Formula I-XV in the
manufacture of a medicament for the treatment of a CNS disorder.
[0046] As used herein, compounds of any of Formula I-XV includes compounds of
Formula I, compounds of Formula II, compounds of Formula III, compounds of
Formula
IV, or compounds of Formula V.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] The features of the present disclosure are set forth with particularity
in the
appended claims. A better understanding of the features and advantages of the
present
invention will be obtained by reference to the following detailed description
that sets forth
illustrative embodiments, in which the principles of the invention are
utilized, and the
accompanying drawings of which:
[0048] Figure 1 describes illustrative shapes of dendritic spines.
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[0049] Figure 2 describes modulation of dendritic spine head diameter by a
small
molecule PAK inhibitor.
[0050] Figure 3 describes modulation of dendritic spine length by a small
molecule
PAK inhibitor.
DETAILED DESCRIPTION OF THE INVENTION
[0051] Provided herein are methods for treatment of CNS conditions by
administration
of inhibitors of certain p21 activated kinases to individuals in need thereof.
Such kinase
inhibitors are inhibitors of one or more of PAK1, PAK2, PAK3, PAK4, PAK5 or
PAK6
kinases. In certain embodiments, the individual has been diagnosed with or is
suspected of
suffering from a CNS disorder such as a neuropsychiatric and/or
neurodegenerative and/or
neurodevelopmental disease or condition that is mediated by p21 activated
kinases. In some
instances, provided herein are methods for treating conditions characterized
by abnormal
dendritic spine morphology and/or spine density and/or spine length and/or
spine thickness
comprising inhibiting PAK activity by administration of a therapeutically
effective amount
of a PAK inhibitor to an individual diagnosed with or suspected of suffering
from a CNS
disorder (e.g., Schizophrenia, Psychotic disorder, schizoaffective disorder,
schizophreniform, Alzheimer's disease, Age-related cognitive decline, Mild
cognitive
impairment, cognitive decline associated with menopause, Parkinson's Disease,
Huntington's Disease, Substance abuse and substance dependence, Fragile X,
Rett's
syndrome, Angelman Syndrome, Asperger's Syndrome, Autism, Autism Spectrum
Disorders, Neurofibromatosis I, Neurofibromatosis H, Tuberous sclerosis,
Clinical
Depression, Bipolar Disorder, Mania, Epilepsy, Mental retardation, Down's
syndrome,
Niemann-Pick disease, Spongiform encephalitis, Lafora disease, Maple syrup
urine disease,
maternal phenylketonuria, atypical phenylketonuria, Generalized Anxiety
Disorder, Turner
Syndrome, Lowe Syndrome, Obsessive-compulsive disorder, Panic disorder,
Phobias,
Posttraumatic Stress Disorder, Anorexia Nervosa, and Bulimia Nervosa).
[0052] A number of CNS disorders are characterized by abnormal dendritic spine
morphology, spine size, spine plasticity and/or spine density as described in
a number of
studies referred to herein. PAK kinase activity has been implicated in spine
morphogenesis,
maturation, and maintenance. See, e.g., Kreis et al (2007), JBiol Chem,
282(29):21497-
21506; Hayashi et al (2007), Proc Natl Acad Sci USA., 104(27):11489-11494,
Hayashi et
al (2004), Neuron, 42(5):773-787; Penzes et al (2003), Neuron, 37:263-274. In
some
embodiments, inhibition or partial inhibition of one or more PAKs normalizes
aberrant
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dendritic spine morphology and/or synaptic function. CNS disorders that are
treated by the
methods described herein include, but are not limited to, Schizophrenia,
Psychotic disorder,
schizoaffective disorder, schizophreniform, Alzheimer's disease, Age-related
cognitive
decline, Mild cognitive impairment, cognitive decline associated with
menopause,
Parkinson's Disease, Huntington's Disease, Substance abuse and substance
dependence,
Fragile X, Rett's syndrome, Angelman Syndrome, Asperger's Syndrome, Autism,
Autism
Spectrum Disorders, Neurofibromatosis I, Neurofibromatosis II, Tuberous
sclerosis,
Clinical Depression, Bipolar Disorder, Mania, Epilepsy, Mental retardation,
Down's
syndrome, Niemann-Pick disease, Spongiform encephalitis, Lafora disease, Maple
syrup
urine disease, maternal phenylketonuria, atypical phenylketonuria, Generalized
Anxiety
Disorder, Obsessive-compulsive disorder, Panic disorder, Phobias,
Posttraumatic Stress
Disorder, Anorexia Nervosa, and Bulimia Nervosa.
[0053] In some instances, CNS disorders are associated with abnormal dendritic
spine
morphology, spine size, spine plasticity, spine motility, spine density and/or
abnormal
synaptic function. In some instances, activation of one or more of PAK1, PAK2,
PAK3,
PAK4, PAK5 and/or PAK6 kinases is implicated in defective spine morphogenesis,
maturation, and maintenance. Described herein are methods for suppressing or
reducing
PAK activity (e.g., by administering a PAK inhibitor for rescue of defects in
spine
morphology, size, plasticity spine motility and/or density) associated with
CNS disorders as
described herein. Accordingly, in some embodiments, the methods described
herein are
used to treat an individual suffering from a CNS disorder wherein the disease
is associated
with abnormal dendritic spine density, spine size, spine plasticity, spine
morphology, spine
plasticity, or spine motility.
[0054] In some embodiments, any inhibitor of one or more p21-activated kinases
described herein reverses or partially reverses defects in dendritic spine
morphology and/or
dendritic spine density and/or synaptic function that are associated with a
CNS disorder. In
some embodiments, modulation of dendritic spine morphology and/or dendritic
spine
density and/or synaptic function alleviates or reverses cognitive impairment
and/or negative
behavioral symptoms (e.g., social withdrawal, anhedonia or the like)
associated with CNS
disorders such as psychiatric conditions. In some embodiments, modulation of
dendritic
spine morphology and/or dendritic spine density and/or synaptic function halts
or delays
progression of cognitive impairment and/or loss of bodily functions associated
with CNS
disorders.
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[0055] In some instances, cellular changes in brain cells contribute to
pathogenesis of a
CNS disorder. In some instances, abnormal dendritic spine density in the brain
contributes
to the pathogenesis of a CNS disorder. In some instances, abnormal dendritic
spine
morphology contributes to the pathogenesis of a CNS disorder. In some
instances, an
abnormal pruning of dendritic spines or synapses during puberty contributes to
the
pathogenesis of a CNS disorder. In some instances, abnormal synaptic function
contributes
to the pathogenesis of a CNS disorder. In some instances, activation of one or
more PAKs is
associated with abnormal dendritic spine density and/or dendritic morphology
and/or
synaptic function and contributes to the pathogenesis of a CNS disorder. In
some instances,
modulation of PAK activity (e.g., attenuation, inhibition or partial
inhibition of PAK
activity) reverses or reduces abnormal dendritic spine morphology and/or
dendritic spine
density and/or synaptic function. In certain embodiments, modulation of
activity of one or
more Group I PAKs (one or more of PAK1, PAK2 and/or PAK3) reverses or reduces
abnormal dendritic spine morphology and/or dendritic spine density and/or
synaptic
function associated with CNS disorders.
[0056] Abnormal dendritic spine morphology and/or density have been found in a
number of CNS disorders as described below. Accordingly, in some embodiments,
the
methods described herein are used to treat an individual suffering from a CNS
disorder that
is associated with abnormal dendritic spine density, spine size, spine
plasticity, spine
morphology, or spine motility. In some embodiments, the methods described
herein are
used to treat an individual suffering from a CNS disorder, such as a psychotic
disorder, as
described in, by way of example, Example 10 and Example 19 herein. Examples of
psychotic disorders include, but are not limited to, schizophrenia,
schizoaffective disorder,
schizophreniform disorder, brief psychotic disorder, delusional disorder,
shared psychotic
disorder (Folie a Deux), substance induced psychosis, and psychosis due to a
general
medical condition. See, e.g., Black et al. (2004), Am JPsychiatry, 161:742-
744; Broadbelt
et al. (2002), Schizophr Res, 58:75-81; Glantz et al. (2000), Arch Gen
Psychiatry 57:65-73;
and Kalus et al. (2000), Neuroreport, 11:3621-3625. In some instances,
aberrant spine
morphogenesis is associated with negative symptoms (e.g., asociality, blunted
affect,
avolition, alogia, anhedonia or dysphoric mood), and/or cognitive impairment
symptomatic
of schizophrenia. In some instances, aberrant spine morphogenesis is
associated with
positive symptoms and behavioral changes (e.g., social withdrawal,
depersonalization, loss
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of appetite, loss of hygiene, delusions, hallucinations, the sense of being
controlled by
outside forces or the like) symptomatic of schizophrenia.
[0057] In some embodiments, the methods described herein are used to treat an
individual suffering from a mood disorder. Examples of mood disorders include,
but are not
limited to, clinical depression as described in, for example, Example 12
herein, bipolar
disorder, cyclothymia, and dysthymia. See, e.g., Hajszan et al (2005), Eur
JNeurosci,
21:1299-1303; Law et al (2004) Am JPsychiatry, 161(10):1848-1855; Norrholm et
al.
(2001), Synapse, 42:151-163; and Rosoklija et al. (2000), Arch Gen Psychiatry,
57:349-
356.
[0058] In some embodiments, the methods described herein are used to treat an
individual suffering from neurodegenerative disorders (e.g., Parkinson's
disease,
Alzheimer's disease (as described in, for example, Example 12 herein) or the
like). See,
e.g., Dickstein et al (2007), Aging Cell, 6:275-284; and Page et al. (2002),
Neuroscience
Letters, 317:37-41. In some embodiments, the methods described herein are used
to treat an
individual suffering from or suspected of having mild cognitive impairment
(MCI). In some
embodiments, the methods described herein are used to halt or delay
progression of mild
cognitive impairment (MCI) to early dementia, mid-stage dementia or late stage
dementia in
an individual suffering from or suspected of having mild cognitive impairment
(MCI). In
some instances, Alzheimer's disease is associated with abnormal dendritic
spine
morphology, spine size, spine plasticity, spine motility, spine density and/or
abnormal
synaptic function. In some instances, soluble Abeta dimers and/or oligomers
increase PAK
kinase activity at the synapse. In some instances, Abeta plaques and/or
insoluble Abeta
aggregates increase PAK kinase activity at the synapse. In some instances,
increased PAK
kinase activity is associated with defective spine morphogenesis, maturation,
and
maintenance. In some instances, PAK inhibitors reverse defects in synaptic
function and
plasticity in a patient diagnosed with Alzheimer's disease before Abeta
plaques can be
detected. In some embodiments, PAK inhibitors reverse defects in synaptic
morphology,
synaptic transmission and/or synaptic plasticity induced by soluble Abeta
dimers and/or
oligomers. In some embodiments, PAK inhibitors reverse defects in synaptic
morphology,
synaptic transmission and/or synaptic plasticity induced by Abeta oligomers
and/or Abeta-
containing plaques.
[0059] In some embodiments, the methods described herein are used to treat an
individual suffering from epilepsy as described in, for example, Example 20
herein. See,
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e.g., Wong (2005), Epilepsy and Behavior, 7:569-577; Swann et al (2000),
Hippocampus,
10:617-625; and Jiang et al (1998), JNeurosci, 18(20):8356-8368.
[0060] In some embodiments, the methods described herein are used to treat an
individual suffering from Parkinson's Disease or Huntington's Disease. See,
e.g., Neely et
al (2007), Neuroscience, 149(2):457-464; Spires et al (2004), Eur JNeurosci,
19:2799-
2807; Klapstein et al (2001), JNeurophysiol, 86:2667-2677; Ferrante et al
(1991), J
Neurosci, 11:3877-3887; and Graveland et al (1985), Science, 227:770-773.
[0061] In some embodiments, the methods described herein are used to treat an
individual suffering from mental retardation, Fragile X syndrome, autism
spectrum
disorders or the like. Examples for Autism spectrum Disorders include, but are
not limited
to, Rett's syndrome, Angelman Syndrome, Asperger's Syndrome, Fragile X
syndrome or
Tuberous sclerosis.
[0062] In some embodiments, the methods described herein are used to treat an
individual suffering from mental retardation. Mental retardation is a disorder
characterized
by significantly impaired cognitive function and deficits in adaptive
behaviors. Mental
retardation is often defined as an Intelligence Quotient (IQ) score of less
than 70. In some
instances, mental retardation is Down's syndrome, Fetal alcohol syndrome,
Klinefelter's
syndrome, congenital hypothyroidism, Williams syndrome, Smith-Lemli-Opitz
syndrome,
Prader-Willi syndrome Phelan-McDermid syndrome, Mowat-Wilson syndrome,
ciliopathy
or Lowe syndrome.
[0063] In some embodiments, the methods described herein are used to treat an
individual suffering from neurofibromatosis. Neurofibromatosis (NF), also
called von
Recklinghaus disease, is an autosomal dominant genetically-inherited disorder
in which the
nerve tissue grows tumors (i.e., neurofibromas, ocular gliomas or the like).
Patients with
NF 1 exhibit a number of different disease symptoms including increased risk
of forming
nervous system tumors and cognitive deficits such as defects in visual-spatial
function,
attention and motor coordination.
[0064] NF is of Type 1 or Type 2. As used herein, NF includes Type 1 NF and
Type 2
NF. In some instances, Type 1 NF is inherited or results from spontaneous
mutation of
neurofibromin. In some instances, NF Type 1 is associated with learning
disabilities in
individuals affected by the disease. In some instances the disease is
associated with a partial
absence seizure disorder. In some instances NF Type 1 is associated with poor
language,
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visual-spatial skills, learning disability (e.g., attention deficit
hyperactivity disorder),
headache, epilepsy or the like.
[0065] Type 2 NF is inherited or results from spontaneous mutation of merlin.
In some
instances, NF Type 2 causes symptoms of hearing loss, tinnitus, headaches,
epilepsy,
cataracts and/or retinal abnormalities, paralysis and/or learning
disabilities. Patients with
NF 1 and NF2 are at increased risk of forming nervous system tumors. In type 1
patients
this includes dermal and plexiform neurofibromas, malignant peripheral nerve
sheath
tumors (MPNST) and other malignant tumors, while type 2 patients may develop
multiple
cranial and spinal tumors.
[0066] In some instances, developmental disability and/or behavioral problems
associated with NF are associated with an abnormality in dendritic spine
morphology and/or
an abnormality in dendritic spine density and/or an abnormality in synaptic
function. In
some instances, an abnormality in dendritic spine morphology and/or dendritic
spine density
and/or synaptic function is associated with activation of p21-activated kinase
(PAK). In
some instances, modulation of PAK activity (e.g., inhibition or partial
inhibition of PAK)
alleviates, reverses or reduces abnormalities in dendritic spine morphology
and/or dendritic
spine density and/or synaptic function thereby reversing or partially
reversing
developmental disability and/or behavioral problems associated with NF. In
some
instances, modulation of PAK activity (e.g., inhibition or partial inhibition
of PAK)
alleviates, reverses or reduces abnormalities in dendritic spine morphology
and/or dendritic
spine density and/or synaptic function thereby reducing occurrence of seizures
in
individuals diagnosed with NF. In some instances, modulation of PAK activity
(e.g.,
inhibition or partial inhibition of PAK) alleviates, reverses or reduces
abnormalities in
dendritic spine morphology and/or dendritic spine density and/or synaptic
function thereby
reducing or reversing learning disabilities associated with NF. In some
instances,
modulation of PAK activity (e.g., inhibition or partial inhibition of PAK)
alleviates,
reverses or reduces cognitive deficits associated with NF. In some instances,
modulation of
PAK activity (e.g., inhibition or partial inhibition of PAK) alleviates,
reverses or reduces
learning disability and/or epilepsy and/or any other symptoms associated with
NF. In some
instances, modulation of PAK activity (e.g., inhibition or partial inhibition
of PAK)
alleviates, reverses or reduces the incidence of tumor development associated
with NF.
[0067] In some embodiments, the methods described herein are used to treat an
individual suffering from Epilepsy, Niemann-Pick disease, spongiform
encephalitis, Lafora
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disease, Maple syrup urine disease, maternal phenylketonuria, atypical
phenylketonuria,
age-related cognitive decline and cognitive decline associated with menopause.
[0068] In some instances, development of a CNS disorder is associated with a
genetic
component. Certain risk alleles and genes that have been identified for CNS
disorders. For
example, for Alzheimer's disease, risk alleles and genes include mutations in
Amyloid
Precursor Protein (APP), mutations in presenilin 1 and 2, the epsilon4 allele,
the 9 lbp allele
in the telomeric region of 12q, Apolipoprotein E-4 (APOE4) gene, SORL1 gene,
reelin gene
or the like. For example, in some instances, development of schizophrenia is
associated with
mutations in the DISCI gene. In some instances, several risk alleles or genes
are involved in
etiology of a CNS disorder. In some instances, CNS disorders run in families
and there is a
predisposition or vulnerability to the illness. In some instances, a
combination of genetic,
familial and environmental factors play a role in manifestation of disease
symptoms. In
some instances, mutations in genes resulting in a predisposition to a CNS
disorders leads to
early-onset of the disease.
Dendritic Spines
[0069] A dendritic spine is a small membranous protrusion from a neuron's
dendrite that
serves as a specialized structure for the formation, maintenance, and/or
function of
synapses. Dendritic spines vary in size and shape. In some instances, spines
have a bulbous
head (the spine head) of varying shape, and a thin neck that connects the head
of the spine
to the shaft of the dendrite. In some instances, spine numbers and shape are
regulated by
physiological and pathological events. In some instances, a dendritic spine
head is a site of
synaptic contact. In some instances, a dendritic spine shaft is a site of
synaptic contact.
Figure 1 shows examples of different shapes of dendritic spines. Dendritic
spines are
"plastic." In other words, spines are dynamic and continually change in shape,
volume, and
number in a highly regulated process. In some instances, spines change in
shape, volume,
length, thickness or number in a few hours. In some instances, spines change
in shape,
volume, length, thickness or number occurs within a few minutes. In some
instances, spines
change in shape, volume, length, thickness or number occurs in response to
synaptic
transmission and/or induction of synaptic plasticity. By way of example,
dendritic spines
are headless (filopodia as shown, for example, in Figure la), thin (for
example, as shown in
Figure lb), stubby (for example as shown in Figure lc), mushroom-shaped (have
door-knob
heads with thick necks, for example as shown in Figure ld), ellipsoid (have
prolate spheroid
heads with thin necks, for example as shown in Figure 1 e), flattened
(flattened heads with
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thin neck, for example as shown in Figure If) or branched (for example as
shown in Figure
1 g).
[0070] In some instances, mature spines have variably-shaped bulbous tips or
heads,
-0.5-2 m in diameter, connected to a parent dendrite by thin stalks 0.1-1 m
long. In
some instances, an immature dendritic spine is filopodia-like, with a length
of 1.5 - 4 m
and no detectable spine head. In some instances, spine density ranges from 1
to 10 spines
per micrometer length of dendrite, and varies with maturational stage of the
spine and/or the
neuronal cell. In some instances, dendritic spine density ranges from 1 to 40
spines per 10
micrometer in medium spiny neurons.
[0071] In some instances, the shape of the dendritic spine head determines
synpatic
function. Defects in dendritic spine morphology and/or function have been
described in
neurological diseases. As an example only, the density of dendritic spines has
been shown
to be reduced in pyramidal neurons from patients with schizophrenia (Glanz and
Lewis,
Arch Gen Psychiatry, 2000:57:65-73). In another example, neurons from patients
with
Fragile X mental retardation show a significant increase in the overall
density of dendritic
spines, together with an increase in the proportion of "immature", filopodia-
like spines and
a corresponding reduction of "mature", mushrooms-shaped spines (Irvin et al,
Cerebral
Cortex, 2000; 10:1038-1044). In many cases, the dendritic spine defects found
in samples
from human brains have been recapitulated in rodent models of the disease and
correlated to
defective synapse function and/or plasticity. In some instances, dendritic
spines with larger
spine head diameter form more stable synapses compared with dendritic spines
with smaller
head diameter. In some instances, a mushroom-shaped spine head is associated
with normal
or partially normal synaptic function. In some instances, a mushroom-shaped
spine is a
healthier spine (e.g., having normal or partially normal synapses) compared to
a spine with
a reduced spine head size, spine head volume and/or spine head diameter. In
some
instances, inhibition or partial inhibition of PAK activity results in an
increase in spine head
diameter and/or spine head volume and/or reduction of spine length, thereby
normalizing or
partially normalizing synaptic function in individuals suffering or suspected
of suffering
from a CNS disorder.
p21-activated kinases (PAKs)
[0072] The PAKs constitute a family of serine-threonine kinases that is
composed of
"conventional", or Group I PAKs, that includes PAK1, PAK2, and PAK3, and "non-
conventional", or Group II PAKs, that includes PAK4, PAKS, and PAK6. See,
e.g., Zhao et
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CA 02776770 2012-04-04
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al. (2005), Biochem J, 386:201-214. These kinases function downstream of the
small
GTPases Rac and/or Cdc42 to regulate multiple cellular functions, including
dendritic
morphogenesis and maintenance (see, e.g., Ethell et al (2005), Prog in
Neurobiol, 75:161-
205; Penzes et al (2003), Neuron, 37:263-274), motility, morphogenesis,
angiogenesis, and
apoptosis, (see, e.g., Bokoch et al., 2003, Annu. Rev. Biochem., 72:743; and
Hofmann et al.,
2004, J. Cell Sci., 117:4343;). GTP-bound Rac and/or Cdc42 bind to inactive
PAK,
releasing steric constraints imposed by a PAK autoinhibitory domain and/or
permitting
PAK phosphorylation and/or activation. Numerous phosphorylation sites have
been
identified that serve as markers for activated PAK.
[0073] In some instances, upstream effectors of PAK include, but are not
limited to,
TrkB receptors; NMDA receptors; adenosine receptors; estrogen receptors;
integrins, EphB
receptors; CDKS, FMRP; Rho-family GTPases, including Cdc42, Rac (including but
not
limited to Racl and Rac2), Chp, TC10, and Wrnch-1; guanine nucleotide exchange
factors
("GEFs"), such as but not limited to GEFT, a-p-2l-activated kinase interacting
exchange
factor (aPIX), Kalirin-7, and Tiaml; G protein-coupled receptor kinase-
interacting protein 1
(GIT1), and sphingosine.
[0074] In some instances, downstream effectors of PAK include, but are not
limited to,
substrates of PAK kinase, such as Myosin light chain kinase (MLCK), regulatory
Myosin
light chain (R-MLC), Myosins I heavy chain, myosin II heavy chain, Myosin VI,
Caldesmon, Desmin, Op18/stathmin, Merlin, Filamin A, LIM kinase (LIMK), Ras,
Raf,
Mek, p47phox, BAD, caspase 3, estrogen and/or progesterone receptors, RhoGEF,
GEF-H1,
NET1, Gaz, phosphoglycerate mutase-B, RhoGDI, prolactin, p4lArc, cortactin
and/or
Aurora-A (See, e.g., Bokoch et al., 2003, Annu. Rev. Biochem., 72:743; and
Hofmann et al.,
2004, J. Cell Sci., 117:4343). Other substances that bind to PAK in cells
include CIB;
sphingolipids; lysophosphatidic acid, G-protein R and/or 7 subunits; PIX/COOL;
GIT/PKL;
Nef; Paxillin; NESH; SH3-containing proteins (e.g. Nck and/or Grb2); kinases
(e.g. Akt,
PDK1, PI 3-kinase/p85, CdkS, Cdc2, Src kinases, Abl, and/or protein kinase A
(PKA));
and/or phosphatases (e.g. phosphatase PP2A, POPX1, and/or POPX2).
PAK inhibitors
[0075] Described herein are PAK inhibitors that treat one or more symptoms
associated
with CNS disorders. Also described herein are pharmaceutical compositions
comprising a
PAK inhibitor (e.g., a PAK inhibitor compound described herein) for reversing
or reducing
one or more of cognitive impairment and/or dementia and/or negative symptoms
and/or
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positive symptoms associated with CNS disorders. Also described herein are
pharmaceutical compositions comprising a PAK inhibitor (e.g., a PAK inhibitor
compound
described herein) for halting or delaying the progression of cognitive
impairment and/or
dementia and/or negative symptoms and/or positive symptoms associated with CNS
disorders. Described herein is the use of a PAK inhibitor for manufacture of a
medicament
for treatment of one or more symptoms of a CNS disorder.
[0076] In some embodiments, the PAK inhibitor is a Group I PAK inhibitor that
inhibits, for example, one or more Group I PAK polypeptides, for example,
PAK1, PAK2,
and/or PAK3. In some embodiments, the PAK inhibitor is a PAK1 inhibitor. In
some
embodiments, the PAK inhibitor is a PAK2 inhibitor. In some embodiments, the
PAK
inhibitor is a PAK3 inhibitor. In some embodiments, the PAK inhibitor is a
mixed
PAK1/PAK3 inhibitor. In some embodiments, the PAK inhibitor is a mixed
PAK1/PAK2
inhibitor. In some embodiments, the PAK inhibitor is a mixed PAK1/PAK4
inhibitor. In
some embodiments, the PAK inhibitor is a mixed PAK1/PAK2/PAK4 inhibitor. In
some
embodiments, the PAK inhibitor is a mixed PAK1/PAK2/PAK3/PAK4 inhibitor. In
some
embodiments, the PAK inhibitor inhibits all three Group I PAK isoforms (PAK1,
2 and
PAK3) with equal or similar potency. In some embodiments, the PAK inhibitor is
a Group
II PAK inhibitor that inhibits one or more Group II PAK polypeptides, for
example PAK4,
PAK5, and/or PAK6. In some embodiments, the PAK inhibitor is a PAK4 inhibitor.
In
some embodiments, the PAK inhibitor is a PAK5 inhibitor. In some embodiments,
the PAK
inhibitor is a PAK6 inhibitor.
[0077] In certain embodiments, a PAK inhibitor described herein reduces or
inhibits the
activity of one or more of PAK1, PAK2, PAK3, and/or PAK4 while not affecting
the
activity of PAK5 and PAK6. In some embodiments, a PAK inhibitor described
herein
reduces or inhibits the activity of one or more of PAK1, PAK2 and/or PAK3
while not
affecting the activity of PAK4, PAK5 and/or PAK6. In some embodiments, a PAK
inhibitor
described herein reduces or inhibits the activity of one or more of PAK1,
PAK2, PAK3,
and/or one or more of PAK4, PAK5 and/or PAK6. In some embodiments, a PAK
inhibitor
described herein is a substantially complete inhibitor of one or more PAKs. As
used herein,
"substantially complete inhibition" means, for example, > 95% inhibition of
one or more
targeted PAKs. In other embodiments, "substantially complete inhibition"
means, for
example, > 90% inhibition of one or more targeted PAKs. In some other
embodiments,
"substantially complete inhibition" means, for example, > 80 % inhibition of
one or more
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targeted PAKs. In some embodiments, a PAK inhibitor described herein is a
partial inhibitor
of one or more PAKs. As used herein, "partial inhibition" means, for example,
between
about 40% to about 60% inhibition of one or more targeted PAKs. In other
embodiments,
"partial inhibition" means, for example, between about 50% to about 70%
inhibition of one
or more targeted PAKs. As used herein, where a PAK inhibitor substantially
inhibits or
partially inhibits the activity of a certain PAK isoform while not affecting
the activity of
another isoform, it means, for example, less than about 10% inhibition of the
non-affected
isoform when the isoform is contacted with the same concentration of the PAK
inhibitor as
the other substantially inhibited or partially inhibited isoforms. In other
instances, where a
PAK inhibitor substantially inhibits or partially inhibits the activity of a
certain PAK
isoform while not affecting the activity of another isoform, it means, for
example, less than
about 5% inhibition of the non-affected isoform when the isoform is contacted
with the
same concentration of the PAK inhibitor as the other substantially inhibited
or partially
inhibited isoforms. In yet other instances, where a PAK inhibitor
substantially inhibits or
partially inhibits the activity of a certain PAK isoform while not affecting
the activity of
another isoform, it means, for example, less than about 1% inhibition of the
non-affected
isoform when the isoform is contacted with the same concentration of the PAK
inhibitor as
the other substantially inhibited or partially inhibited isoforms.
[0078] Provided herein, in certain embodiments, are compounds having the
structure of
Formula I or pharmaceutically acceptable salt or N-oxide thereof:
R7
N
(R5)r B
N N N 0
H I
Q
Formula I;
wherein:
(R4)s
R7 is " R3
wherein ring T is an aryl, or a heteroaryl ring;
R3 is a substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted
heteroaryl attached to ring T via a carbon atom of R3, or a substituted or
unsubstituted heterocycloalkyl attached to ring T via a carbon atom of R3;
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Q is a substituted or unsubstituted alkyl, a substituted or unsubstituted
heteroalkyl, a
substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted
cycloalkyl, a substituted or unsubstituted cycloalkylalkyl, a substituted or
unsubstituted heterocycloalkylalkyl, a substituted or unsubstituted aryl, a
substituted or unsubstituted arylalkyl, a substituted or unsubstituted
heteroaryl,
or a substituted or unsubstituted heteroarylalkyl;
each R4 is independently halogen, -CN, -NO2, -OH, -OCF3, -OCH2F, -OCF2H, -CF3,
-SR8, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -OC(=O)R9, -C02R10,
-N(R10)2, -C(=O)N(R10)2, -NR10C(=O)R10, -N R10C(=O)OR1O,
-NR10C(=O)N(R10)2, a substituted or unsubstituted alkyl, a substituted or
unsubstituted alkoxy, a substituted or unsubstituted heteroalkyl, a
substituted or
unsubstituted cycloalkyl, or a substituted or unsubstituted heterocycloalkyl;
R8 is H or R9;
R9 is a substituted or unsubstituted alkyl, a substituted or unsubstituted
cycloalkyl, a
substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted
aryl,
or a substituted or unsubstituted heteroaryl;
each R10 is independently H, a substituted or unsubstituted alkyl, a
substituted or
unsubstituted cycloalkyl, a substituted or unsubstituted heterocycloalkyl, a
substituted or unsubstituted aryl, or a substituted or unsubstituted
heteroaryl; or
two R10, together with the atoms to which they are attached form a
heterocycle;
ring B is aryl or heteroaryl;
each R5 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -S(=O)2R9,
NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -OC(=O)R9, -C02R10, -N(R10)2, -
C =0 N R10 NR10C(=O)R10 NR10C(=O)OR1O NR10C(=O)N(R10)2, -OR10, a
substituted or unsubstituted alkyl, a substituted or unsubstituted alkoxy, a
substituted or unsubstituted heteroalkyl, a substituted or unsubstituted
cycloalkyl, or a substituted or unsubstituted heterocycloalkyl;
r is 0 to 8; and
s is 0 to 4.
[0079] In one embodiment is a compound of Formula I wherein ring T is an aryl
ring.
In one embodiment, the aryl ring is a phenyl group. In another embodiment is a
compound
of Formula I wherein ring T is a heteroaryl ring. In yet another embodiment is
a compound
of Formula I, wherein ring T is selected from pyrrole, furan, thiophene,
pyrazole, imidazole,
23
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isoxazole, oxazole, isothiazole, thiazole, 1,2,3-triazole, 1,3,4-triazole, 1-
oxa-2,3-diazole, 1-
oxa-2,4-diazole, 1-oxa-2,5-diazole, 1-oxa-3,4-diazole, 1-thia-2,3-diazole, 1-
thia-2,4-diazole,
1-thia-2,5-diazole, 1-thia-3,4-diazole, tetrazole, pyridine, pyridazine,
pyrimidine, and
pyrazine. In another embodiment, ring T is thiazole.
[0080] In a further embodiment is a compound of Formula I, wherein R3 is a C-
linked
heterocycloalkyl. In one embodiment, the C-linked heterocycloalkyl is oxetane,
azetidine,
tetrahydrofuran, pyrrolidine, tetrahydrothiophene, piperidine,
tetrahydropyran, and
morpholine. In a further embodiment, the C-linked heterocycloalkyl is
substituted with at
least one Ci-C6alkyl or halogen. In another embodiment, the Ci-C6alkyl is
methyl, ethyl, or
n-propyl. In one embodiment is a compound of Formula I, wherein R3 is a
substituted or
unsubstituted C-linked heteroaryl. In one embodiment, R3 is selected from a C-
linked
pyrrole, furan, thiophene, pyrazole, imidazole, isoxazole, oxazole,
isothiazole, thiazole,
1,2,3-triazole, 1,3,4-triazole, 1-oxa-2,3-diazole, 1-oxa-2,4-diazole, 1-oxa-
2,5-diazole, 1-oxa-
3,4-diazole, 1-thia-2,3-diazole, 1-thia-2,4-diazole, 1-thia-2,5-diazole, 1-
thia-3,4-diazole,
tetrazole, pyridine, pyridazine, pyrimidine, and pyrazine. In yet another
embodiment, R3 is
a C-linked thiazole. In another embodiment, R3 is a C-linked pyrazole. In a
further
embodiment, R3 is a C-linked oxadiazole. In another embodiment, R3 is a
substituted or
unsubstituted cycloalkyl. In a further embodiment, cycloalkyl is selected from
cyclopropyl,
cyclobutyl, cyclopentyl, or cyclohexyl. In a further embodiment, R3 is
cyclopentyl. In
another embodiment, R3 is cyclohexyl.
[0081] In yet another embodiment, R3 is a C-linked heteroaryl substituted with
at least
one group selected from halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -S(=O)2R9,
NR10S =0 2R9 S =0 2N R10 C(=O)R8 OC(=O)R9 C02R'0 N(Ri0)2, -C(=O)N(R10)2,
-NR10C(=O)R10, -NR10C(=O)OR10, -NR10C(=O)N(R10)2, -OR10, a substituted or
unsubstituted alkyl, a substituted or unsubstituted alkoxy, a substituted or
unsubstituted
heteroalkyl, a substituted or unsubstituted cycloalkyl, or a substituted or
unsubstituted
heterocycloalkyl. In one embodiment, the C-linked heteroaryl is substituted
with Ci-
C6alkyl. In another embodiment, Ci-C6alkyl is methyl, ethyl, n-propyl, iso-
propyl, n-butyl,
iso-butyl, or tert-butyl. In a further embodiment, the C-linked heteroaryl is
substituted with
methyl. In another embodiment, ethyl. In a further embodiment, n-propyl or iso-
propyl.
[0082] Also disclosed herein is a compound of Formula I wherein R4 is
independently
halogen, -CN, -NO2, -OH, -OCF3, -OCH2F, -OCF2H, -CF3, -SRB, -NR1OS(=0)2R9,
-S(=O)2N(R10)2, -C(=O)R9, -OC(=O)R8, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
24
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NR10C(=O)R1O, -N R10C(=O)OR1O, and -NR10C(=O)N(R10)2. In a further embodiment,
R4 is
a halogen. In yet another embodiment, R4 is selected from F, Cl, Br, or I. In
another
embodiment, R4 is F. In yet another embodiment, R4 is a substituted or
unsubstituted alkyl,
a substituted or unsubstituted alkoxy, a substituted or unsubstituted
heteroalkyl, a
substituted or unsubstituted cycloalkyl, or a substituted or unsubstituted
heterocycloalkyl.
In one embodiment, R4 is substituted or unsubstituted alkyl selected from
methyl, ethyl, n-
propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In another embodiment,
R4 is OH. In a
further embodiment, R4 is OCH3. In yet another embodiment, R4 is OCF3.
[0083] In another embodiment, s is 1. In yet another embodiment, s is 0.
[0084] In one embodiment, is a compound of Formula I wherein Q is a
substituted or
unsubstituted alkyl, a substituted or unsubstituted heteroalkyl, a substituted
or unsubstituted
heterocycloalkyl, a substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted
cycloalkylalkyl, a substituted or unsubstituted heterocycloalkylalkyl, a
substituted or
unsubstituted aryl, a substituted or unsubstituted arylalkyl, a substituted or
unsubstituted
heteroaryl, or a substituted or unsubstituted heteroarylalkyl. In another
embodiment, Q is a
substituted or unsubstituted alkyl. In a further embodiment, Q is an
unsubstituted methyl,
ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl or tert-butyl. In a further
embodiment, Q is
ethyl.
[0085] In yet another embodiment, is a compound of Formula I, wherein ring B
is an
aryl ring. In another embodiment, ring B is a substituted or unsubstituted
phenyl. In a
further embodiment, ring B is a substituted or unsubstituted naphthalene. In a
further
embodiment, is a compound of Formula I, wherein ring B is a heteroaryl ring
selected from
pyrrole, furan, thiophene, pyrazole, imidazole, isoxazole, oxazole,
isothiazole, thiazole,
1,2,3-triazole, 1,3,4-triazole, 1-oxa-2,3-diazole, 1-oxa-2,4-diazole, 1-oxa-
2,5-diazole, 1-oxa-
3,4-diazole, 1-thia-2,3-diazole, 1-thia-2,4-diazole, 1-thia-2,5-diazole, 1-
thia-3,4-diazole,
tetrazole, pyridine, pyridazine, pyrimidine, and pyrazine.
[0086] In yet a further embodiment, is a compound of Formula I, wherein R5 is
a C3-C6
cycloalkyl ring; or a 3-6-membered heterocycloalkyl ring comprising 1-3 N
atoms, an 0
atom, a S atom; or any combination thereof, and wherein R5 is further
substituted by
halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -S(=O)2R9, NR10S(=0)2R9, -
S(=0)2N(R10)2 -
C(=O)R8, -OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -NR10C(=O)R10,
-NR10C(=O)OR10, -NR10C(=O)N(R10)2, -OR10, substituted or unsubstituted alkyl,
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substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl,
substituted or
unsubstituted cycloalkyl or substituted or unsubstituted heterocycloalkyl.
[0087] In one embodiment, R5 is a C3-C6cycloalkyl ring. In another embodiment,
the
C3-C6cycloalkyl ring is cyclopropyl. In another embodiment, the C3-
C6cycloalkyl ring is
cyclopentyl. In another embodiment, the C3-C6cycloalkyl is cyclohexyl.
[0088] In another embodiment, R5 is OH or CN. In a further embodiment, R5 is
OCF3,
or C173-
[0089] In yet another embodiment is a compound of Formula I wherein r is 0. In
another embodiment, r is 1. In a further embodiment, r is 2.
H
SN,
[0090] In one embodiment is a compound of Formula I wherein (R ) is
selected from:
26
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H
H H H Nl H
0 0'1/
01, 11 N 0 01
s s s s
CN CN `N `N CN CKP
H , H , , , H , A
H H H
Nr+ N sf'S H N
I
/ HNl ~ I \\ N HN I /
N ~/\% O
N H HN F I H
H
N N N N I/ N
N HN J::: O
HN N
H~ HNJ F N H \ NH
.
ss'' H /
I ,
N N Na iN I/ HNO
HNJ F H HN\J F H H
`~ 71
Nj, H H
N P'
N / O I / H N~ I / N
Na I \
HN J CI N H N Oja
H H
N H N HN I\ N H N I \\ N'
OL Z
rN N O~\%
HNJ HN H H
H N
N H H
/ Na ~\ N.~+ \ N.s~
^N I / s'' HNa I / s'
HN HN \ N O
H
F F F H N,~ H \ N N
,s
I\ N _Na N I/ I/
/ H H N a H
H N H ::p NN/
H NO
HN I I N H
H N.s'
N
(and
N O
H
[0091] In one embodiment, is a compound of Formula I, wherein R5 is halogen, -
CN, -
OH, substituted or unsubstituted alkyl, -OR10, -NR10S(=O)2R9, -S(=O)2N(R10)2-
N(R10)2-
C(=O)N(R10)2, -NR10C(=O)R10, -NR10C(=O)OR10, -NR10C(=O)N(R10)2, or substituted
or
27
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unsubstituted heterocycloalkyl. In one embodiment, R5 is selected from F, Cl,
Br, or I. In
another embodiment R5 is F.
[0092] In another embodiment, is a compound of Formula I, wherein at least one
R5 is -
NR10S(=O)2R9, -S(=O)2N(R10)2, -N(R1O)2, -C(=O)N(R10)2, -NR10C(=O)R10,
-NR10C(=O)OR1O, -NR10C(=O)N(R10)2, or substituted or unsubstituted
heterocycloalkyl. In
one embodiment, is a compound of Formula I, wherein at least one R5 is -
N(R10)2, or
substituted or unsubstituted heterocycloalkyl. In yet another embodiment, is a
compound of
Formula I wherein at least one of R5 is a substituted or unsubstituted
piperazine, substituted
or unsubstituted piperidine, substituted or unsubstituted pyrrolidine or
substituted or
unsubstituted morpholine. In a further embodiment, is a compound of Formula I,
wherein at
least one R5 is -OR10. In one embodiment is a compound of Formula I, wherein
at least one
R5 is -OR10 and Rio is H. In another embodiment, Rio is alkyl selected from
methyl, ethyl,
n-propyl, iso-propyl, n-butyl, iso-butyl, and tert-butyl.
[0093] In one embodiment is a compound of Formula I wherein ring B is
substituted
with N(R10)2, wherein R10 is each independently selected from H and a
substituted or
unsubstituted heterocycloalkyl. In another embodiment is a compound of Formula
I
wherein ring B is substituted with -NHR10 wherein Rio is a substituted or
unsubstituted
piperazine, substituted or unsubstituted piperidine, substituted or
unsubstituted pyrrolidine
or substituted or unsubstituted morpholine. In a further embodiment is a
compound of
Formula I wherein ring B is substituted with N(CH3)R10 wherein Rio is a
substituted or
unsubstituted piperazine, substituted or unsubstituted piperidine, substituted
or
unsubstituted pyrrolidine or substituted or unsubstituted morpholine.
[0094] Also presented herein is a compound of Formula I wherein ring B is
substituted
with -OR10 wherein Rio is a substituted or unsubstituted heterocycloalkyl. In
another
embodiment is a compound of Formula I wherein ring B is substituted with -OR10
wherein
R10 is a substituted or unsubstituted piperazine, substituted or unsubstituted
piperidine,
substituted or unsubstituted pyrrolidine or substituted or unsubstituted
morpholine. In yet
another embodiment is a compound of Formula I wherein ring B is substituted
with at least
one CF3.
[0095] In yet another embodiment, ring B is substituted with at least two R5.
In another
embodiment, ring B is substituted with halogen and a substituted or
unsubstituted
heterocycloalkyl. In another embodiment, ring B is substituted with at least
one F, Cl, Br,
28
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or I and a substituted or unsubstituted piperazine, substituted or
unsubstituted piperidine,
substituted or unsubstituted pyrrolidine, or substituted or unsubstituted
morpholine.
[0096] In another aspect is a compound having the structure of Formula II or
pharmaceutically acceptable salt or N-oxide thereof:
R3
T
~~ N (R4 )s
(R5)r \ 11
`~ H
Q
Formula II;
wherein:
ring T is an aryl, or a heteroaryl ring;
R3 is a substituted or unsubstituted cycloalkyl, a substituted or
unsubstituted heteroaryl
attached to ring T via a carbon atom of R3, or a substituted or unsubstituted
heterocycloalkyl attached to ring T via a carbon atom of R3;
each R4 is independently halogen, -CN, -NO2, -OH, -OCF3, -OCF2H, -CF3, -SR8, -
S(=O)R9, -S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -OR10, -C(=O)R8, -
OC(=O)R9, -CO2R10, -N(R10)2, -C(=O)N(R10)2, -NR10C(=O)R10, -N R10C(=O)OR10,
-NR10C(=O)N(R10)2, substituted or unsubstituted alkyl, substituted or
unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl or substituted or
unsubstituted
heterocycloalkyl;
R8 is H or R9;
R9 is a substituted or unsubstituted alkyl, a substituted or unsubstituted
cycloalkyl, a
substituted or unsubstituted heterocycloalkyl, a substituted or unsubstituted
aryl, or a
substituted or unsubstituted heteroaryl;
each R10 is independently H, substituted or unsubstituted alkyl, substituted
or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,
substituted
or unsubstituted aryl or substituted or unsubstituted heteroaryl, or two R10
together
with the atoms to which they are attached form a heterocycle;
s is 0-4;
ring B is aryl or heteroaryl;
each R5 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -S(=O)2R9,
NR10S =0 2R9 S =0 2N R10 C(=O)R8 OC(=O)R9 CO2R10 N(R10)2 -
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C =0 N R10 NR10C(=O)R10 NR10C(=O)OR10 NR10C(=O)N(R10)2, -OR10,
substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl; and
r is 0-8.
[0097] In a further embodiment is a compound having the structure of Formula
III:
R4 (R4)s1
T
N \ \ R3
(R5)r
N N N O
H
Formula III;
wherein s 1 is 0 to 3 and ring T, ring B, R3, R4, R5, Q and r are described
previously.
[0098] In yet a further embodiment is a compound having the structure of
Formula IV:
R3
(R4)s
N
(R5)r
N N N O
H
Formula IV;
wherein s 1 is 0 to 4 and ring B, R3, R4, R5, Q and r are described
previously.
[0099] In another embodiment is a compound having the structure of Formula V:
R3
N \ \
(R5 )r
N N N O
H
Formula V;
wherein s 1 is 0 to 4 and ring B, R3, R4, R5, Q and r are described
previously.
[00100] In another embodiment is a compound having the structure of Formula
Va:
R3
(R4)s1
N
(R5)r
N N N O
H
Formula Va;
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wherein s 1 is 0 to 4 and ring B, R3, R4, R5, Q and r are described
previously.
[00101] In another embodiment is a compound having the structure of Formula
Vb:
R4 R3
N
N
(R5), N N N O
H
Formula Vb;
wherein ring B, R3, R4, R5, Q and r are described previously.
[00102] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb
wherein R3 is selected from pyrrole, furan, thiophene, pyrazole, imidazole,
isoxazole,
oxazole, isothiazole, thiazole, 1,2,3-triazole, 1,3,4-triazole, 1-oxa-2,3-
diazole, 1-oxa-2,4-
diazole, 1-oxa-2,5-diazole, 1-oxa-3,4-diazole, 1-thia-2,3-diazole, 1-thia-2,4-
diazole, 1-thia-
2,5-diazole, 1-thia-3,4-diazole, tetrazole, pyridine, pyridazine, pyrimidine,
and pyrazine.
[00103] In another embodiment, is a compound of Formula I, II, III, IV, V, Va,
or Vb
wherein R3 is selected from
31
CA 02776770 2012-04-04
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N~ N/> N' 0 N
NON N's/ N NI ~NN
I ~~ I ~N I I S ~ yN N N
,
0 S p S /N7N-N , N-N
sss' I N I \N I I \N I I \N i' O
iN N' N
N , N N
~'CO ~N H H ~-CNH
N
i N D, ""CN N H N -/ ~O
N / N-S
s' ~ s' ss'3 p ~ D
ND O O N ~/ = I`' O 0 `O
N
0
~O N and N n
and
S-O
O
0
[00104] In a further embodiment is a compound of Formula I, II, III, IV, V,
Va, or Vb,
wherein (R5)r-@ is:
32
CA 02776770 2012-04-04
WO 2011/044535 PCT/US2010/052106
\ \ 0 f `T N f `T N,
(R5)r I / (R5)r i / / (R5)r / (R5)r I / / (R5)r i / N
f `T N r `T N f T O~ rr Ss (N Y N
(R5)r li 1N (R5)r LI /1 /> (R5)r N (R5)r (R
N \~ `N ,
N__'N 5 rN (N (NN 5 rNYN
(R )r I / (R )r N / (R5), (R5), (R )r N
N N
(NN, 5 N mi l ' N' 5 N, NYS
(R5)r N (R )r / N (R )r N (R5)r II I N (R5)r
N
NS fN~ \ \ \ i \ \
(R5)r N (R5)r / / (R5)r N (R5)r or (R5)r
N N
[00105] In another embodiment is a compound of Formula I, II, III, IV, V, Va,
or Vb,
where R5 is halogen, -CN, -OH, a substituted or unsubstituted alkyl, -OR10, -
NR10S(=O)2R9,
-S(=O)2N(R10)2, -N(R10)2, -C(=O)N(R10)2, -NR10C(=O)R10, -NR10C(=O)OR10,
5 -NR10C(=O)N(R10)2, or a substituted or unsubstituted heterocycloalkyl.
[00106] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein at least one R5 is -NR10S(=O)2R9, -S(=O)2N(R10)2, -N(R10)2, -
C(=O)N(R10)2, -
NR10C(=O)R10, -NR10C(=O)OR10, -NR10C(=O)N(R10)2, or a substituted or
unsubstituted
heterocycloalkyl.
[00107] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein at least one R5 is -N(R10)2, or a substituted or unsubstituted
heterocycloalkyl. In a
further embodiment is a compound of Formula I, II, III, IV, V, Va, or Vb
wherein at least
one of R5 is a substituted or unsubstituted piperazine, a substituted or
unsubstituted
piperidine, a substituted or unsubstituted pyrrolidine, or a substituted or
unsubstituted
morpholine. In one embodiment is a compound of Formula I, II, III, IV, V, Va,
or Vb,
wherein at least one R5 is -OR10. In another embodiment is a compound of
Formula I, II,
III, IV, V, Va, or Vb, wherein R4 is independently halogen, -CN, -OH, -OCF3, -
OCF3, -
OCF2H, -CF3, -SR8, a substituted or unsubstituted alkyl, or a substituted or
unsubstituted
alkoxy.
[00108] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein s is zero.
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[00109] In a further embodiment is a compound of Formula I, II, III, IV, V,
Va, or Vb,
wherein Q is a substituted or unsubstituted alkyl, or a substituted or
unsubstituted
heteroalkyl. In another embodiment is a compound of Formula I, II, III, IV, V,
Va, or Vb,
wherein Q is a substituted or unsubstituted cycloalkyl, or a substituted or
unsubstituted
heterocycloalkyl. In a further embodiment is a compound of Formula I, II, III,
IV, V, Va, or
Vb, wherein Q is a substituted or unsubstituted cycloalkylalkyl, or a
substituted or
unsubstituted heterocycloalkylalkyl. In one embodiment is a compound of
Formula I, II,
III, IV, V, Va, or Vb, wherein Q is a substituted or unsubstituted aryl, or a
substituted or
unsubstituted heteroaryl.
[00110] In one embodiment is a compound of Formula I, II, III, IV, V, Va, or
Vb,
wherein Q is a substituted or unsubstituted arylalkyl, or a substituted or
unsubstituted
heteroarylalkyl.
[00111] In another embodiment is a compound of Formula I, II, III, IV or V,
wherein Q
is selected from:
F N ~,N.
IS
O
O O N CN)
O=1S" O
O
\ \ N~s O
and
F NI1 N
F
[00112] Also provided herein, in some embodiments, are compounds having the
structure
of Formula VI or pharmaceutically acceptable salt or N-oxide thereof:
R6
R7
N
(R5)r &N"~~N N O
H I
WN-1
Q
Formula VI
34
CA 02776770 2012-04-04
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wherein:
W is a bond;
R6 is -CN, -OH, substituted or unsubstituted alkoxy, -N(R10)2, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted aryl or substituted or unsubstituted
heteroaryl;
R7 is halogen, -CN, -OH, substituted or unsubstituted alkoxy, -
C(=O)N(R10)2, -CO2R10, -N(R10)2, acyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or
substituted or unsubstituted heteroaryl;
Q is substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkylalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl, or
substituted or unsubstituted cycloalkyl or heterocycloalkyl fused to ring
A;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -CO2R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -N R10C(=O)OR10, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
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each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
with the atoms to which they are attached form
a heterocycle;
ring B is aryl or heteroaryl substituted with R5;
each R5 is independently halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R1O, -NR10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
[00113] In one embodiment, is a compound having the structure of Formula VI or
pharmaceutically acceptable salt or N-oxide thereof wherein:
W is a bond;
R6 is -CN, -OH, substituted or unsubstituted alkoxy, -N(R10)2, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted aryl or substituted or unsubstituted
heteroaryl;
R7 is halogen, -CN, -OH, substituted or unsubstituted alkoxy, -
C(=O)N(R10)2, -C02R10, -N(R10)2, acyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or
substituted or unsubstituted heteroaryl;
Q substituted or unsubstituted heteroalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkylalkyl, substituted or unsubstituted
heterocycloalkylalkyl, substituted or unsubstituted aryl, substituted or
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unsubstituted arylalkyl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted heteroarylalkyl;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -N R10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
with the atoms to which they are attached form
a heterocycle;
ring B is aryl or heteroaryl substituted with R5;
each R5 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R1O, -NR10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
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[00114] In another embodiment is a compound of structure of Formula VI or
pharmaceutically acceptable salt or N-oxide thereof wherein:
W is a bond;
R6 is -CN, -OH, substituted or unsubstituted alkoxy, -N(R10)2, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted aryl or substituted or unsubstituted
heteroaryl;
R7 is halogen, -CN, -OH, substituted or unsubstituted alkoxy, -
C(=O)N(R10)2, -C02R10, -N(R10)2, acyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or
substituted or unsubstituted heteroaryl;
Q is an unsubstituted alkyl;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R1O, -N R10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
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with the atoms to which they are attached form
a heterocycle;
ring B is aryl or heteroaryl substituted with R5;
each R5 is independently halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -CO2R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -NR10C(=O)OR10, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
[00115] In yet another embodiment is a compound of structure of Formula VI or
pharmaceutically acceptable salt or N-oxide thereof wherein:
W is a bond;
R6 is -CN, -OH, substituted or unsubstituted alkoxy, -N(R10)2, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted aryl or substituted or unsubstituted
heteroaryl;
R7 is halogen, -CN, -OH, substituted or unsubstituted alkoxy, -
C(=O)N(R10)2, -CO2R10, -N(R10)2, acyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or
substituted or unsubstituted heteroaryl;
Q is a substituted alkyl;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -CO2R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -N R10C(=O)OR10, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
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unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
with the atoms to which they are attached form
a heterocycle;
ring B is aryl or heteroaryl substituted with R5;
each R5 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R1O, -NR10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
[00116] Provided herein, in some embodiments, are compounds having the
structure
of Formula VII or pharmaceutically acceptable salt or N-oxide thereof:
R6
-Iz:~ R7
N ~
(R5)r B
N N N O
H I
WNI-I
Q
Formula VII
CA 02776770 2012-04-04
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wherein:
W is a bond;
R6 is substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted aryl or substituted or
unsubstituted heteroaryl;
R7 is H, halogen, -CN, -OH, substituted or unsubstituted alkyl, substituted or
unsubstituted alkoxy, -C(=O)N(R10)2, -C02R10, -N(R10)2, acyl,
substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted aryl or substituted or unsubstituted heteroaryl;
Q is substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkylalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R1O, -N R10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
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unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
with the atoms to which they are attached form
a heterocycle;
ring B is aryl or heteroaryl substituted with R5;
each R5 is independently halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R1O, -NR10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
[00117] In one embodiment is a compound of Formula VII wherein Q is
substituted
or unsubstituted alkyl. In a further embodiment is a compound of Formula VII
wherein
Q is a substituted alkyl. In yet another embodiment is a compound of Formula
VII
wherein Q is an unsubstituted alkyl. In a further embodiment is a compound of
Formula
VII wherein Q is substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkylalkyl,
substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted
heteroaryl, substituted or unsubstituted heteroarylalkyl.
[00118] Provided herein, in some embodiments, are compounds having the
structure
of Formula VIII or pharmaceutically acceptable salt or N-oxide thereof:
R6
R7
(R5)r B
N N N O
H I
WN-1
Q
Formula VIII
wherein:
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W is a bond;
R6 is H, or halogen;
R7 is acyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl or
substituted or unsubstituted heteroaryl;
Q is substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkylalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R1O, -N R10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
with the atoms to which they are attached form
a heterocycle;
ring B is aryl or heteroaryl substituted with R5;
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each R5 is independently halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R9, -
OC(=O)R8, -CO2R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -NR10C(=O)OR10, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
[00119] In one embodiment is a compound of Formula VIII wherein Q is
substituted
or unsubstituted alkyl. In a further embodiment is a compound of Formula VIII
wherein
Q is a substituted alkyl. In yet another embodiment is a compound of Formula
VIII
wherein Q is an unsubstituted alkyl. In a further embodiment is a compound of
Formula
VIII wherein Q is substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkylalkyl,
substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted
heteroaryl, substituted or unsubstituted heteroarylalkyl.
[00120] Also provided herein, in some embodiments, are compounds having the
structure of Formula IX or pharmaceutically acceptable salt or N-oxide
thereof:
R6
R7
N
(R5),
N N N 0
H I
WN
Q
Formula IX
wherein:
W is a bond;
R6 is substituted or unsubstituted alkyl;
R7 is substituted or unsubstituted heteroalkyl, substituted or unsubstituted
cycloalkyl, or substituted or unsubstituted heterocycloalkyl;
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Q is substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkylalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -N R10C(=O)OR10, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
with the atoms to which they are attached form
a heterocycle;
ring B is aryl or heteroaryl substituted with R5;
each R5 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -NR10C(=O)OR1O, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
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alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
[00121] In one embodiment is a compound of Formula IX wherein Q is substituted
or
unsubstituted alkyl. In a further embodiment is a compound of Formula IX
wherein Q
is a substituted alkyl. In yet another embodiment is a compound of Formula IX
wherein
Q is an unsubstituted alkyl. In a further embodiment is a compound of Formula
IX
wherein Q is substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkylalkyl,
substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted
heteroaryl, substituted or unsubstituted heteroarylalkyl.
[00122] Provided herein, in some embodiments, are compounds having the
structure
of Formula X or pharmaceutically acceptable salt or N-oxide thereof:
R6
R7
~ ~
(R5)r B
N N N O
H I
WN-1
Q
Formula X
wherein:
W is a bond;
R6 is H;
R7 is
(R4)s
'X( W
ring T is aryl, heteroaryl, cycloalkyl or heterocycloalkyl substituted with R3
and
R4.
R3 is a substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted cycloalkyl or substituted or
unsubstituted heterocycloalkyl attached to ring T via a carbon atom;
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Q is substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkylalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted heteroarylalkyl;
ring A is substituted or unsubstituted aryl or heteroaryl substituted with 0-4
R4;
each R4 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, -NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -N R10C(=O)OR10, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
R8 is H or substituted or unsubstituted alkyl;
R9 is substituted or unsubstituted alkyl, substituted
or unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl
each R10 is independently H, substituted or
unsubstituted alkyl, substituted or
unsubstituted cycloalkyl, substituted or
unsubstituted aryl or substituted or
unsubstituted heteroaryl, or two R10 together
with the atoms to which they are attached form
a heterocycle;
s is 0-4;
ring B is aryl or heteroaryl substituted with R5;
each R5 is independently halogen, -CN, -NO2, -OH, -SR8, -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -C02R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -NR10C(=O)OR1O, -NR10C(=O)N(R10)2,
47
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substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8.
[00123] In one embodiment is a compound of Formula X wherein Q is substituted
or
unsubstituted alkyl. In a further embodiment is a compound of Formula X
wherein Q is
a substituted alkyl. In yet another embodiment is a compound of Formula X
wherein Q
is an unsubstituted alkyl. In a further embodiment is a compound of Formula X
wherein
Q is substituted or unsubstituted heteroalkyl, substituted or unsubstituted
heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkylalkyl,
substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted
heteroaryl, substituted or unsubstituted heteroarylalkyl.
[00124] Provided herein, in some embodiments, are compounds having the
structure
of Formula XI or pharmaceutically acceptable salt or N-oxide thereof:
R6
R7
~ ~
(R5)r B
N N N O
H I
WN-1
Q
Formula XI
wherein:
W is N-Ria;
Rla is H or substituted or unsubstituted alkyl;
Q is substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylalkyl,
substituted or unsubstituted heterocycloalkylalkyl, substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted heteroaryl, or substituted or unsubstituted heteroarylalkyl;
ring B is aryl or heteroaryl substituted with R5;
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each R5 is independently halogen, -CN, -NO2, -OH, -SR', -S(=O)R9, -
S(=O)2R9, NR10S(=O)2R9, -S(=O)2N(R10)2, -C(=O)R8, -
OC(=O)R9, -CO2R10, -N(R10)2, -C(=O)N(R10)2, -
NR10C(=O)R10, -NR10C(=O)OR10, -NR10C(=O)N(R10)2,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkoxy, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl or substituted or unsubstituted
heterocycloalkyl;
r is 0-8;
R6 is H, halogen, -CN, -OH, substituted or unsubstituted alkyl, substituted or
unsubstituted alkoxy, substituted or unsubstituted heteroalkyl, -N(R10)2,
substituted or unsubstituted cycloalkyl, substituted or unsubstituted aryl or
substituted or unsubstituted heteroaryl;
R7 is H, halogen, -CN, -OH, acyl, substituted or unsubstituted alkyl,
substituted
or unsubstituted alkoxy, -C(=O)N(R10)2, -CO2R10, -N(R10)2, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted
or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or
substituted or unsubstituted heteroaryl.
[00125] In one embodiment is a compound of Formula XI wherein Q is substituted
or
unsubstituted alkyl. In a further embodiment is a compound of Formula XI
wherein Q
is a substituted alkyl. In yet another embodiment is a compound of Formula XI
wherein
Q is an unsubstituted alkyl. In a further embodiment is a compound of Formula
XI
wherein Q is substituted or unsubstituted heteroalkyl, substituted or
unsubstituted
heterocycloalkyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted
cycloalkylalkyl, substituted or unsubstituted heterocycloalkylalkyl,
substituted or
unsubstituted aryl, substituted or unsubstituted arylalkyl, substituted or
unsubstituted
heteroaryl, substituted or unsubstituted heteroarylalkyl.
[00126] In a further aspect is a compound having the structure of Formula XII:
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Ft
~~ N \ \
N N N O
H I
Y3- N
QA
Y6
(R4)s
Formula XII
wherein:
each of Y3, Y4 and Y5 are independently N-Rla, CR1R2, SO2, or C=O;
Rla is H or substituted or unsubstituted alkyl;
R1 and R2 are each independently H or substituted or unsubstituted alkyl.
[00127] In some embodiments, a compound of Formula XI has the structure of
formula XIII:
(R5)r \ is /~ ~ i
H N O
N
R1 ar"
(R4)s
Formula XIII
[00128] In some embodiments, a compound of Formula XI has the structure of
formula XIV:
R6
(R 5~
H N i O (R4)s
A
R1a N
P
R1 R2
Formula XIV
wherein:
p is 1, 2 or 3;
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R1and R2 are each independently H or substituted or unsubstituted alkyl; or R1
and R2
together with the carbon to which they are attached form a C3-C6 cycloalkyl
ring.
[00129] In some embodiments of Formula XVI, ring A is a heteroaryl ring. In
some
embodiments of Formula XVI, ring A is an aryl ring. In some embodiments of
Formula
XVI, ring A is a heterocycloalkyl ring. In some embodiments of Formula XVI,
ring A is
a cycloalkyl ring.
[00130] In some embodiments, the compound of Formula XI has the structure of
Formula XV:
R3
R7
N
N N N O
H
Y3, N
YX
(R4)s
Formula XV
wherein:
each of Y3, Y4 and Y5 are independently N-RIa, CR1R2, SO2, or C=O;
Rla is H or substituted or unsubstituted alkyl;
R1 and R2 are each independently H or substituted or unsubstituted alkyl.
[00131] In some embodiments, the compound of Formula XI has the structure of
Formula XVA, Formula XVB, Formula XVC or Formula XVD:
R6 R6
R7 N R
N 5
(R5)r~R II (R )r i
N O
N O H
H
N
N
(R11)r- (R11)k
(R4), (R4)s
Formula XVA Formula XVB
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R6
R6 f : R7
Nom'' N N N io
(RI), --OB " I 't, H
N (RI j,)
k
(R11)Ec~ err
(R4)s (84)8
Formula X .VC Formula X 'I)
wherein:
each R' is independently fl, halogen, substituted or unsubstituted alkyl,
substituted or unsubstituted al.koxy. or two R" together with the carbon
atom to which they are attached form c=o; and
k is 1-4.
1001321 In a further aspect is a compound having the structure:
CA CA
1. ,K
4 r~ rV
IANII-IAII
,õ..
52
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F^i :~r. .<{per ~. ~\ U \N ~ ~
rt#tr ~ ~~^~~
t, ~ ~ r \ p
N / c
~ F ~.,fa. = 4 F 1
rr t rr~.ry R, a ~xra
t.t Lrr=
FF
~1ba~ :fi,r;++~~Ft
53
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r3 ~ fit'
16,
icy f' .r,, a fp cE r " 'tt tQ 'y
,i~+1 37~~. t3 _
r ~ t~ c
o" N
tt
t,' k~ vhf c3. f N:! Y:.`
~tir~ +~ ra' '~o # tom, ~.~ ~ ~=~,, ~~ 1,~~ f,~,"y c=r~ -~
KI-I F ` r=fir - ie "E w.
54
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"'r F
I e~ ~ r ~
2 =k ? - r` Pt
rr "'~ ry >~ a= ^.r=! t: tFS `~Cr ~"'n# r, rY t
.~
ra'=
. -=,~. C,~ # -. w e,;t'~=`eF ~~ 1:.fia T~ jt!'~r NTC T
i kkY
`~ 2=Y't+f CF t7 ..N.~:a='J['-f '..^-=PJ IN ff;_
k-h
~N``'r, N ~i ~:=f' " tf C-1-Fa -=*' ~'F F o~-Fi
5 EA
}
CA 02776770 2012-04-04
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71,
". Mme`"~ Ci } ~ rY- tt~ N'M "`~ t 1' ~ M Cd C
Pd M
41-
~~` P=3 ?~d ~. li4~ra' w~xe :31
S ti
5to
CA 02776770 2012-04-04
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I /-N
I=-
t_.~~ tt rF. J .~ r x k1-1 rr' hr~ s
ri,Y ! c
Ilk,
fY,
+ _ 2 \, #
1 k f l 1. L
AV"
41
`~ et1'`rs3 1 slrte f , `:, r rrlb
I;r
X F
lti. 14. ~.
Pr r:~
57
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~, +.v ;= \1 ~t1 l' .ia.~ of to ~~`
\ 7 r^.
jx"
Pte,.
,-- r -fl !7
kts rr=xc,{., k = }"ljt', fpj,.\~ af, ~`. t Rte} =1$ F
t"r pp rJ
CA 02776770 2012-04-04
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aa. S h1
-`kaõ \ L d iii N
-l r ~. ~`i.~~ ` ` f~n~ t'ax,,.'"e'Y `p~= 7, f T~c` Lll~.
r:~,= . Mp ~.~ ~. ~. 1 ~ ~'^ ra.~, P.~.+. ~.et~~ .~E Ra t,~~: rf `'ca a
r ~, r
E 4- t t -'~ ra"~y~"a,..,~,~~1,. ~ l h. ; =~
~`t=rJ'}`h' .C. FIe rr"~ ,'`s't
rl
`..' C LC ht s .t .`Y. K4- r a r : 9. kyy~#'L~g, y,, i
'ZVI
::fit r= .
"= \ ~ ~'dd rn
CA 02776770 2012-04-04
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,{Y 'Ma{p.'y~ \:.,.rr\ .iC~gxr, 2 Vr7'i.l~f." t-`-
..t~.'=õa...ty~
Z X
f ~W
r: Z'V: N `!V" \ rte,? -.~'=N
r ra x :r rs
=5, YY
"Al
i 1 rF t
:'Y'=
Pr,('uY'U'71
lk~ N,-1- 1A 'All
i) ri rr F4 j tr
Y~.r
CA 02776770 2012-04-04
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t, r
+~~ t ~d x, rt~t ^. T3`~ ,Y'\i^,/`~.PI ~ ~ t=t~~~y~-Y&
\ ` +.~ t=~ ~,~` Vii. ? i V =.
a r
1
x-N
r ~~~ r~ `rlfy.
, rl ^4
~'--
=J
61
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rj
4-k lk tell 'All
Lkrt r,
C+'= F
F= N
iC , ,^=, 11,., t... a-'`~ N,r rte:' f~-`~ 39~~.~ !d~ N
11 t-L~
Al.
N t of
ia'Kerr c '~r'r` ``r:r
d
^...=~.rl
rf
62
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y~'~'rl r; t, ~. Cry . Nt
hl,
t is
F. te
-"sa <
J 44
Pi \-N
o gg 7[
rr et ~
r~ v rA ~. N y , f
63
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7 ~T~ r.~; = ~' t iF J
fie
~t L
rr r'~ f=l~'f:
s { ~, [L -Al
rr ra' ~
yrr~i K=~`td--{ ~> rr-~-~ r, r~-~ mow'{- ~"
^'.`1.~ 'r1 hd` L.=. '4,,. \ ~'~~K~tJ~~~< ti.`s=1 ,,=~rw a ~~C,
l tt fl " E
64
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\oa;. /`.~ ? `'fit 1.1 t `~ ~
rJ` [ 'All r-r rc= r.F ,. v f " rr'
<.' i r S EE~ r f
Y
f0l
rN;.r
L ~ 't 1
a r ` k, rt¾
It :~ l Z
:q4 >-. N~~ p~ .us\~ f C r'r: 7 , ~ q~1tfr
I~j
t+(
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'.T `= r~ ,~`v lr .F Vsr }r *?, P.I~`~..'~
G:s
7 S
N }'
~4 ..S
t c t r.-. r~ ,,=
1 ri
,= ~ ', t if ~ 1
f t
3 s s
.. ItJ...~. r <
I~A
66
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~+ ~tez zV ~t rt" t'' ra.
_` N g
f4l
td
Fk R ci Yi !
~i.N.
~= Z, .tl h .~ ~,
r1 r
^~h
67
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ILI
N
N HN,-\. G1 \ .ter t ` G
N
I i
N
l\ J
N
N '0
H N N N 0
H
J, b
N~ I ~ Y HN
I i E
.~. \ i
N
N N N" Ed N N
r r
H H i
N N N \ 4 N N N%-`0
H H
N , N =\
HN
N
N N N 0 H
H N
^: CEO t `:. ;~ ? Ny AÃ
HN 1 F r 't N N N'~ z~,
N
N N 0
H
1 ;t3
C9 10
H# N N/~~
N \i N NN 0
N,^ H
N N N 0
H
~N. .0 N N. N N ' . ~ N- ftiil ~N
~\rN\ = N N-err N
N N N CÃ
N N N ..0
H
I\`
68
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t 9fi`1
rlw N . r
.E
\:
r.~. ` `~_ ~ =~} rte' \r Sl _, =.>r="~,r, ~~' t ]t-r~:~rr-~
a v,_3
r\ F 1.
trk& 'tfrFf'
..(`= F.#~ N ~= { rat' ~ x
69
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r\"" q '=':~`~"~,.~ ri ~ N`..J c ~ ~ ~ i+. ~.---~ ~~~ Nr `'~ ~ fem.
Yf
ht` Y F
_fw r3
t1' ' r r k
If I
t4~ r- K'N r r'"i a
AS`y 'Y' t J Y (!.^ r i ~ ~.I! rFY,I.~45..
l.rY~ ter'
rk~
fib
`rte
~'.
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I.~rr.3 =:.:j I% N
hr"` i to"~ rt 41
nr.. Q
4-1
r N r
r fi r ~
MtV '
'Ilk
41-
5
,ell
71
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ht N
t ,~ rf
}
Jt r1 'tom i .'~ i._f it T$pvtj( _`a
M'J
Er \,
~.. ->\t~ \~ pp It ~ .N \ ~'w F i `f `t ~^~~ J'4 k{ aY,,ar i
yyyQQQ~~~ N-N
66 ~
h
7 22
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t\~'\~rr .N c= E
14
iI l,g
CF
ra \ ~ ~ Tom,:..
q. Al
C C~kw~t ~F
F.. n,~ =rl
--ram ~ ft~f
Lr ~
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'XX
t --r>
Ct (~N ~C
to r ti-.w= t. i r.i i - \ w "Y- '.
t 1 ~\
iu_-;s~~;~.r,~r~,= ~ ~`".l,~gt'`''lrsFfF\t,Ft ._ ~" ~ t\ ,~ f~~4. L ,y
\ Pd' N= ~G+
l ~s= K.
_ ~;' ~" ~. ,rte ,,=~-=.. a
N ~.
l
rD~
74
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t t;
tJ=~,f=N
:Nz
S +~
ll ~~
Ihl 4 ems" Fj
melt"
Irk wk~., (q Ata -f
a=ti,tF ~ M1;N
rs-
r ~'r~ rat r^'_~
\,1. rJ' r 15,: rt., rah-.N
n 4 rr~` a"` ' c~ A A tt rpp~ 3 I
.~t'~r'rJr tJ c:< ~n~tr;. ~ ~.~*~~;rti r+~ rJty
rs r
7S
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fk E ` 'N
}
vF=\; XI
k3'x;.} t i ~'N 1' Nay
~,i3 r~ t r rt` F.t `_-~ rt{~ CX'?-t
t t ~!.
or a pharmaceutically acceptable salt, solvate or N -oxide
thereof.
1001.33] In some embodiments, a PAK :inhibitor is a small molecule. As
referred to
herein.. a "small molecule" is an organic molecule that is less than about 5
kilodaltons (kDa)
in size. In. some embodiments, the small molecule is less than about 4 kiDa.,
3 kiDa, about 2
kl)a, or about l kDa, In some embodiments, the small molecule is less than
about 800
-10 daltons (Da), about 600 Da, about 500 Da, about 400 Da, about 00 Da. about
200 Da, or
about 100 Da. In some embodiments, a small molecule is less than about 4000
g/mol, less
than about 3000ghnol, 2000 g/nnol, loss than about 1500 g.!mol, less than
about 1000 gimol,
less than about 800 g/luol,or less than about 500 g/mol. In sonic embodiments,
small
molecules are non-polymeric. F's picall t small molecules are not proteins,
polypeptidcs,
1.5 polynucleotides, oli o nucleotides, polysaccharides, glycoprotei as, or
proteoglycans, but
includes peptides of up to about 40 amino acids. A derivative of a small
molecule refers to a
molecule that shares the same structural core as the original. small.
molecule, but which is
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CA 02776770 2012-04-04
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prepared by a series of chemical reactions from the original small molecule.
As one
example, a pro-drug of a small. molecule is a derivative of that small
molecule.An analog of
a small molecule refers to a molecule that shares the same or similar
structural core as the
original small molecule, and which is synthesized by a similar or related
route, or art-
recognized variation, as the original small molecule.
1001341 In certain embodiments, compounds described herein. have one or more
chiral
centers. As such, all stereoisorers are envisioned herein. In various
embodiments,
compounds described herein are present in optically active or racemic forms.
It is tobe
understood that the compounds described herein encompass racemic, optically-
active,
1.0 regioisonaeric and stereoisomeric forms, or combinations thereof that
possess the
therapeutically useful. properties described herein. Preparation of optically
active forms is
achieve in any suitable manner. including by way of non-limiting example, by
resolution of
the racemic form by recrystallization techniques, by, synthesis from optically-
active starting
materials, by chiral synthesis, or by chromatographic separation using a
chiral stationary
t phase. In some embodiments, mixtures of one or more isomer is utilized as
the therapeutic
compound described herein. In certain embodiments, compounds described herein
contains
one or more chiral centers. These compounds are prepared by any means,
including
enantioselective synthesis and/or separation of a mixture of enantiomers
and/or
diastereomers. Resolution of compounds and isomers thereof is achieved by any
means
2t including, by way of non-limiting example, chemical processes, enzymatic
processes,
fractional crystallization, distillation, chromatography, and the litre.
(001351 In various embodiments, pharmaceutically acceptablesalts described
herein
include, by way of non-limiting example, a nitrate, chloride, bromide,
phosphate, sulfate,
acetate, hexafluorophosphate, citrate, gluconate, benzoate propionate,
butyrate,
25 sulfosilicylate, maleate, laurate, malate, f aanarate. s acc.inate,
tartrate, amsonate, parnoate, p-
tcaluiietiesullanate, mesylate and the like. l urther oie, pharmaceutically
acceptable salts
include, by way of non-limiting example, alkaline earth metal salts (e.g..
calcium or
rna nesiuan , alkali metal salts (e.g., sodium-dependerit or potassium),
ammonium salts and
the like.
30 [001361 Compounds described herein also include isotopically-labeled
compounds
wherein one or more atoms is replaced by an atom having the same atomic
number, but an
atomic mass or mass number different from the atomic mass or mass number
usually found
in nature. Examples of isotopes suitable for inclusion in the compounds
described herein
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include and are not limited to 2H 3H, 11C, 13C, 14C, 36CI, isF, 1231, 1251,
13N, 15N, 150,170
, ,
110, 32P 35S or the like. In some embodiments, isotopically-labeled compounds
are useful in
drug and/or substrate tissue distribution studies. In some embodiments,
substitution with
heavier isotopes such as deuterium affords certain therapeutic advantages
resulting from
greater metabolic stability (for example, increased in vivo half-life or
reduced dosage
requirements). In some embodiments, substitution with positron emitting
isotopes, such as
11C 18F 150 and 13N, is useful in Positron Emission Topography (PET) studies
for
examining substrate receptor occupancy. Isotopically-labeled compounds are
prepared by
any suitable method or by processes using an appropriate isotopically-labeled
reagent in
place of the non-labeled reagent otherwise employed.
[00137] The compounds described herein, and other related compounds having
different
substituents are synthesized using techniques and materials described herein
and as
described, for example, in Fieser and Fieser's Reagents for Organic Synthesis,
Volumes 1-
17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes
1-5
and Supplementals (Elsevier Science Publishers, 1989); Organic Reactions,
Volumes 1-40
(John Wiley and Sons, 1991), Larock's Comprehensive Organic Transformations
(VCH
Publishers Inc., 1989), March, ADVANCED ORGANIC CHEMISTRY 4th Ed., (Wiley
1992);
Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4th Ed., Vols. A and B (Plenum
2000, 2001), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3rd
Ed.,
(Wiley 1999) (all of which are incorporated by reference for such disclosure).
General
methods for the preparation of compound as described herein are modified by
the use of
appropriate reagents and conditions, for the introduction of the various
moieties found in the
formula as provided herein. As a guide the following synthetic methods are
utilized.
[00138] Compounds described herein are synthesized using any suitable
procedures
starting from compounds that are available from commercial sources, or are
prepared using
procedures described herein.
Formation of Covalent Linkages by Reaction of an Electrophile with a
Nucleophile
[00139] The compounds described herein are modified using various
electrophiles and/or
nucleophiles to form new functional groups or substituents. Table A entitled
"Examples of
Covalent Linkages and Precursors Thereof' lists selected non-limiting examples
of covalent
linkages and precursor functional groups which yield the covalent linkages.
Table A is used
as guidance toward the variety of electrophiles and nucleophiles combinations
available that
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provide covalent linkages. Precursor functional groups are shown as
electrophilic groups
and nucleophilic groups.
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Table A: Examples of Covalent Linkages and Precursors Thereof
Covalciit Linkage product Electrophile Nucleophilc
Carboxamides Activated esters amines/anilines
Carboxamides acyl azides amines/anilines
Carboxamides acyl halides amines/anilines
Esters acyl halides alcohols/phenols
Esters acyl nitriles alcohols/phenols
Carboxamides acyl nitriles amines/anilines
Imines Aldehydes amines/anilines
Hydrazones aldehydes or ketones Hydrazines
Oximes aldehydes or ketones Hydroxylamines
lkyl amines alkyl halides amines/anilines
Esters alkyl halides carboxylic acids
Thioethers alkyl halides Thiols
Ethers alkyl halides alcohols/phenols
Thioethers alkyl sulfonates Thiols
Esters alkyl sulfonates carboxylic acids
Ethers alkyl sulfonates alcohols/phenols
Esters Anhydrides alcohols/phenols
Carboxamides Anhydrides amines/anilines
Thiophenols aryl halides Thiols
Aryl amines aryl halides mines
Thioethers zindines Thiols
Boronate esters Boronates Glycols
Carboxamides carboxylic acids amines/anilines
Esters carboxylic acids Alcohols
ydrazines Hydrazides carboxylic acids
-acylureas or Anhydrides carbodiimides carboxylic acids
Esters diazoalkanes carboxylic acids
Thioethers Epoxides Thiols
Thioethers aloacetamides Thiols
mmotriazines alotriazines amines/anilines
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Covalent Linkage Product Electropliile Nuclcopliilc
Triazinyl ethers alotriazines alcohols/phenols
midines imido esters amines/anilines
reas Isocyanates amines/anilines
Urethanes Isocyanates alcohols/phenols
Thioureas isothiocyanates amines/anilines
Thioethers Maleimides Thiols
Phosphite esters hosphoramidites Alcohols
Silyl ethers silyl halides Alcohols
Alkyl amines sulfonate esters amines/anilines
Thioethers sulfonate esters Thiols
Esters sulfonate esters carboxylic acids
Ethers sulfonate esters Alcohols
Sulfonamides sulfonyl halides amines/anilines
Sulfonate esters sulfonyl halides phenols/alcohols
Use of Protecting Groups
[00140] In the reactions described, it is necessary to protect reactive
functional groups,
for example hydroxy, amino, imino, thio or carboxy groups, where these are
desired in the
final product, in order to avoid their unwanted participation in reactions.
Protecting groups
are used to block some or all of the reactive moieties and prevent such groups
from
participating in chemical reactions until the protective group is removed. In
some
embodiments it is contemplated that each protective group be removable by a
different
means. Protective groups that are cleaved under totally disparate reaction
conditions fulfill
the requirement of differential removal.
[00141] In some embodiments, protective groups are removed by acid, base,
reducing
conditions (such as, for example, hydrogenolysis), and/or oxidative
conditions. Groups such
as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are acid labile
and are used to
protect carboxy and hydroxy reactive moieties in the presence of amino groups
protected
with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which
are base
labile. Carboxylic acid and hydroxy reactive moieties are blocked with base
labile groups
such as, but not limited to, methyl, ethyl, and acetyl in the presence of
amines blocked with
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acid labile groups such as t-butyl carbamate or with carbamates that are both
acid and base
stable but hydrolytically removable.
[00142] In some embodiments carboxylic acid and hydroxy reactive moieties are
blocked
with hydrolytically removable protective groups such as the benzyl group,
while amine
groups capable of hydrogen bonding with acids are blocked with base labile
groups such as
Fmoc. Carboxylic acid reactive moieties are protected by conversion to simple
ester
compounds as exemplified herein, which include conversion to alkyl esters, or
are blocked
with oxidatively-removable protective groups such as 2,4-dimethoxybenzyl,
while co-
existing amino groups are blocked with fluoride labile silyl carbamates.
[00143] Allyl blocking groups are useful in the presence of acid- and base-
protecting
groups since the former are stable and are subsequently removed by metal or pi-
acid
catalysts. For example, an allyl-blocked carboxylic acid is deprotected with a
Pd -catalyzed
reaction in the presence of acid labile t-butyl carbamate or base-labile
acetate amine
protecting groups. Yet another form of protecting group is a resin to which a
compound or
intermediate is attached. As long as the residue is attached to the resin,
that functional group
is blocked and does not react. Once released from the resin, the functional
group is available
to react.
[00144] Typically blocking/protecting groups are selected from: 0
HZ ~s HZ
F
HZC=C'CZ \ I C\ \ I O HZCIC\CZ oH3C-\
O
allyl Bn Cbz alloc Me
H2 H2 0
CH3 / v \
H C (H3C)3C' H3Cisl Si
3 (H3C)3C (CH3)3C O
Et t-butyl TBDMS Teoc 0
H2C-O_11/
(CH3)3C (C 6H5)3C- H3C~ \ ~.s \ \
~0 C_~ I
O H3CO c~
Boc PMB trityl acetyl Fmoc
[00145] Other protecting groups, plus a detailed description of techniques
applicable to
the creation of protecting groups and their removal are described in Greene
and Wuts,
Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York,
NY, 1999,
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and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are
incorporated herein by reference for such disclosure.
Certain Definitions
[00146] As used herein the term "Treatment", "treat", or "treating" includes
achieving a
therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit is
meant to include
eradication or amelioration of the underlying disorder or condition being
treated. For
example, in an individual with Huntington's disease, therapeutic benefit
includes alleviation
or partial and/or complete halting of the progression of the disease, or
partial or complete
reversal of the disease. Also, a therapeutic benefit is achieved with the
eradication or
amelioration of one or more of the physiological or psychological symptoms
associated
with the underlying condition such that an improvement is observed in the
patient,
notwithstanding the fact that the patient is still affected by the condition.
For example, in an
individual suffering from epilepsy, therapeutic benefit includes alleviation
or partial and/or
complete halting of seizures, or reduction in frequency of seizures. A
prophylactic benefit of
treatment includes prevention of a condition, retarding the progress of a
condition, or
decreasing the likelihood of occurrence of a condition. As used herein,
"treat", "treating" or
"treatment" includes prophylaxis.
[00147] As used herein, the phrase "abnormal spine size" refers to dendritic
spine
volumes or dendritic spine surface areas (e.g., volumes or surface areas of
the spine heads
and/or spine necks) associated with CNS disorders that deviate significantly
relative to
spine volumes or surface areas in the same brain region (e.g., the CAI region,
the prefrontal
cortex) in a normal individual (e.g., a mouse, rat, or human) of the same age;
such
abnormalities are determined as appropriate, by methods including, e.g.,
tissue samples,
relevant animal models, post-mortem analyses, or other model systems.
[00148] The phrase "defective spine morphology" or "abnormal spine morphology"
or
"aberrant spine morphology" refers to abnormal dendritic spine shapes,
volumes, surface
areas, length, width (e.g., diameter of the neck), spine head diameter, spine
head volume,
spine head surface area, spine density, ratio of mature to immature spines,
ratio of spine
volume to spine length, or the like that is associated with a CNS disorder
relative to the
dendritic spine shapes, volumes, surface areas, length, width (e.g., diameter
of the neck),
spine density, ratio of mature to immature spines, ratio of spine volume to
spine length, or
the like observed in the same brain region in a normal individual (e.g., a
mouse, rat, or
human) of the same age; such abnormalities or defects are determined as
appropriate, by
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methods including, e.g., tissue samples, relevant animal models, post-mortem
analyses, or
other model systems.
[00149] The phrase "abnormal spine function" or "defective spine function" or
"aberrant
spine function" refers to a defect of dendritic spines to undergo stimulus-
dependent
morphological or functional changes (e.g., following activation of AMPA and/or
NMDA
receptors, LTP, LTD, etc) associated with CNS disorders as compared to
dendritic spines in
the same brain region in a normal individual of the same age. The "defect" in
spine function
includes, e.g., a reduction in dendritic spine plasticity, (e.g., an
abnormally small change in
dendritic spine morphology or actin re-arrangement in the dendritic spine), or
an excess
level of dendritic plasticity, (e.g., an abnormally large change in dendritic
spine morphology
or actin re-arrangement in the dendritic spine). Such abnormalities or defects
are determined
as appropriate, by methods including, e.g., tissue samples, relevant animal
models, post-
mortem analyses, or other model systems.
[00150] The phrase "abnormal spine motility" refers to a significant low or
high
movement of dendritic spines associated with a CNS disorder as compared to
dendritic
spines in the same brain region in a normal individual of the same age. Any
defect in spine
morphology (e.g., spine length, density or the like) or synaptic plasticity or
synaptic
function (e.g., LTP, LTD or the like) or spine motility occurs in any region
of the brain,
including, for example, the frontal cortex, the hippocampus, the amygdala, the
CAI region,
the prefrontal cortex or the like. Such abnormalities or defects are
determined as
appropriate, by methods including, e.g., tissue samples, relevant animal
models, post-
mortem analyses, or other model systems.
[00151] As used herein, the phrase "biologically active" refers to a
characteristic of any
substance that has activity in a biological system and/or organism. For
instance, a substance
that, when administered to an organism, has a biological effect on that
organism is
considered to be biologically active. In particular embodiments, where a
protein or
polypeptide is biologically active, a portion of that protein or polypeptide
that shares at least
one biological activity of the protein or polypeptide is typically referred to
as a "biologically
active" portion.
[00152] As described herein, a CNS disorder is a disorder that can affect
either the spinal
cord or brain. By way of example only, CNS disorder include Schizophrenia,
Psychotic
disorder, schizoaffective disorder, schizophreniform, Alzheimer's disease, Age-
related
cognitive decline, Mild cognitive impairment, cognitive decline associated
with menopause,
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Parkinson's Disease, Huntington's Disease, Substance abuse and substance
dependence,
Fragile X, Rett's syndrome, Angelman Syndrome, Asperger's Syndrome, Autism,
Autism
Spectrum Disorders, Neurofibromatosis I, Neurofibromatosis II, Tuberous
sclerosis,
Clinical Depression, Bipolar Disorder, Mania, Epilepsy, Mental retardation,
Down's
syndrome, Niemann-Pick disease, Spongiform encephalitis, Lafora disease, Maple
syrup
urine disease, maternal phenylketonuria, atypical phenylketonuria, Generalized
Anxiety
Disorder, Turner Syndrome, Lowe Syndrome, Obsessive-compulsive disorder, Panic
disorder, Phobias, Posttraumatic Stress Disorder, Anorexia Nervosa, and
Bulimia Nervosa.
[00153] As used herein, Mental retardation is a disorder characterized by
significantly
impaired cognitive function and deficits in adaptive behaviors. By way of
example only,
mental retardation is Down's syndrome, Fetal alcohol syndrome, Klinefelter's
syndrome,
congenital hypothyroidism, Williams syndrome, Smith-Lemli-Opitz syndrome,
Prader-Willi
syndrome Phelan-McDermid syndrome, Mowat-Wilson syndrome, ciliopathy or Lowe
syndrome.
[00154] As used herein, the term "subcortical dementia" refers to symptoms
related to
Huntington's disease (e.g., deficits in executive functions such as planning,
cognitive
flexibility, abstract thinking, rule acquisition, initiating appropriate
actions, inhibiting
inappropriate actions; memory deficits such as short-term memory deficits,
long-term
memory difficulties, deficits in episodic (memory of one's life), procedural
(memory of the
body of how to perform an activity) and working memory, and the like). In some
instances,
"progression toward dementia" is identified, monitored or diagnosed by
neuropsychological
or behavioral testing. In other instances, "progression toward dementia" is
identified,
monitored or diagnosed by neuroimaging or brain scans.
[00155] As used herein, the term "effective amount" is an amount, which when
administered systemically, is sufficient to effect beneficial or desired
results, such as
beneficial or desired clinical results, or enhanced cognition, memory, mood,
or other desired
effects. An effective amount is also an amount that produces a prophylactic
effect, e.g., an
amount that delays, reduces, or eliminates the appearance of a pathological or
undesired
condition associated with a CNS disorder. An effective amount is optionally
administered in
one or more administrations. In terms of treatment, an "effective amount" of a
composition
described herein is an amount that is sufficient to palliate, alleviate,
ameliorate, stabilize,
reverse or slow the progression of a CNS disorder e.g., cognitive decline
toward dementia,
mental retardation or the like. An "effective amount" includes any PAK
inhibitor used alone
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or in conjunction with one or more agents used to treat a disease or disorder.
An "effective
amount" of a therapeutic agent as described herein will be determined by a
patient's
attending physician or other medical care provider. Factors which influence
what a
therapeutically effective amount will be include, the absorption profile
(e.g., its rate of
uptake into the brain) of the PAK inhibitor, time elapsed since the initiation
of disease, and
the age, physical condition, existence of other disease states, and
nutritional status of an
individual being treated. Additionally, other medication the patient is
receiving, e.g.,
antidepressant drugs used in combination with a PAK inhibitor, will typically
affect the
determination of the therapeutically effective amount of the therapeutic agent
to be
administered.
[00156] As used herein, the term "inhibitor" refers to a molecule which is
capable of
inhibiting (including partially inhibiting or allosteric inhibition) one or
more of the
biological activities of a target molecule, e.g., a p21-activated kinase.
Inhibitors, for
example, act by reducing or suppressing the activity of a target molecule
and/or reducing or
suppressing signal transduction. In some embodiments, a PAK inhibitor
described herein
causes substantially complete inhibition of one or more PAKs. In some
embodiments, the
phrase "partial inhibitor" refers to a molecule which can induce a partial
response for
example, by partially reducing or suppressing the activity of a target
molecule and/or
partially reducing or suppressing signal transduction. In some instances, a
partial inhibitor
mimics the spatial arrangement, electronic properties, or some other
physicochemical and/or
biological property of the inhibitor. In some instances, in the presence of
elevated levels of
an inhibitor, a partial inhibitor competes with the inhibitor for occupancy of
the target
molecule and provides a reduction in efficacy, relative to the inhibitor
alone. In some
embodiments, a PAK inhibitor described herein is a partial inhibitor of one or
more PAKs.
In some embodiments, a PAK inhibitor described herein is an allosteric
modulator of PAK.
In some embodiments, a PAK inhibitor described herein blocks the p21 binding
domain of
PAK. In some embodiments, a PAK inhibitor described herein blocks the ATP
binding site
of PAK. In some embodiments, a PAK inhibitor is a "Type II" kinase inhibitor.
In some
embodiment a PAK inhibitor stabilizes PAK in its inactive conformation. In
some
embodiments, a PAK inhibitor stabilizes the "DFG-out" conformation of PAK.
[00157] In some embodiments, PAK inhibitors reduce, abolish, and/or remove the
binding between PAK and at least one of its natural binding partners (e.g.,
Cdc42 or Rac).
In some instances, binding between PAK and at least one of its natural binding
partners is
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stronger in the absence of a PAK inhibitor (by e.g., 90%, 80%, 70%, 60%, 50%,
40%, 30%
or 20%) than in the presence of a PAK inhibitor. Alternatively or
additionally, PAK
inhibitors inhibit the phosphotransferase activity of PAK, e.g., by binding
directly to the
catalytic site or by altering the conformation of PAK such that the catalytic
site becomes
inaccessible to substrates. In some embodiments, PAK inhibitors inhibit the
ability of PAK
to phosphorylate at least one of its target substrates, e.g., LIM kinase 1
(LIMK1), myosin
light chain kinase (MLCK), cortactin; or itself. PAK inhibitors include
inorganic and/or
organic compounds.
[00158] In some embodiments, PAK inhibitors described herein increase
dendritic spine
length. In some embodiments, PAK inhibitors described herein decrease
dendritic spine
length. In some embodiments, PAK inhibitors described herein increase
dendritic neck
diameter. In some embodiments, PAK inhibitors described herein decrease
dendritic neck
diameter. In some embodiments, PAK inhibitors described herein increase
dendritic spine
head diameter. In some embodiments, PAK inhibitors described herein decrease
dendritic
spine head diameter. In some embodiments, PAK inhibitors described herein
increase
dendritic spine head volume. In some embodiments, PAK inhibitors described
herein
decrease dendritic spine head volume. In some embodiments, PAK inhibitors
described
herein increase dendritic spine surface area. In some embodiments, PAK
inhibitors
described herein decrease dendritic spine surface area. In some embodiments,
PAK
inhibitors described herein increase dendritic spine density. In some
embodiments, PAK
inhibitors described herein decrease dendritic spine density. In some
embodiments, PAK
inhibitors described herein increase the number of mushroom shaped spines. In
some
embodiments, PAK inhibitors described herein decrease the number of mushroom
shaped
spines.
[00159] In some embodiments, a PAK inhibitor suitable for the methods
described herein
is a direct PAK inhibitor. In some embodiments, a PAK inhibitor suitable for
the methods
described herein is an indirect PAK inhibitor. In some embodiments, a PAK
inhibitor
suitable for the methods described herein decreases PAK activity relative to a
basal level of
PAK activity by about 1.1 fold to about 100 fold, e.g., to about 1.2 fold, 1.5
fold, 1.6 fold,
1.7 fold, 2.0 fold, 3.0 fold, 5.0 fold, 6.0 fold, 7.0 fold, 8.5 fold, 9.7
fold, 10 fold, 12 fold, 14
fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 90 fold,
95 fold, or by any
other amount from about 1.1 fold to about 100 fold relative to basal PAK
activity. In some
embodiments, the PAK inhibitor is a reversible PAK inhibitor. In other
embodiments, the
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PAK inhibitor is an irreversible PAK inhibitor. Direct PAK inhibitors are
optionally used
for the manufacture of a medicament for treating a CNS disorder.
[00160] In some embodiments, a PAK inhibitor used for the methods described
herein
has in vitro ED50 for PAK activation of less than 100 M (e.g., less than 10
M, less than 5
M, less than 4 M, less than 3 M, less than 1 M, less than 0.8 M, less than
0.6 M, less
than 0.5 M, less than 0.4 M, less than 0.3 M, less than less than 0.2 M,
less than 0.1
M, less than 0.08 M, less than 0.06 M, less than 0.05 M, less than 0.04 M,
less than
0.03 M, less than less than 0.02 M, less than 0.01 M, less than 0.0099 M,
less than
0.0098 M, less than 0.0097 M, less than 0.0096 M, less than 0.0095 M, less
than
0.0094 M, less than 0.0093 M, less than 0.00092 M, or less than 0.0090 M).
[00161] In some embodiments, a PAK inhibitor used for the methods described
herein
has in vitro ED50 for PAK activation of less than 100 M (e.g., less than 10
M, less than 5
M, less than 4 M, less than 3 M, less than 1 M, less than 0.8 M, less than
0.6 M, less
than 0.5 M, less than 0.4 M, less than 0.3 M, less than less than 0.2 M,
less than 0.1
M, less than 0.08 M, less than 0.06 M, less than 0.05 M, less than 0.04 M,
less than
0.03 M, less than less than 0.02 M, less than 0.01 M, less than 0.0099 M,
less than
0.0098 M, less than 0.0097 M, less than 0.0096 M, less than 0.0095 M, less
than
0.0094 M, less than 0.0093 M, less than 0.00092 M, or less than 0.0090 M).
[00162] As used herein, synaptic function refers to synaptic transmission
and/or synaptic
plasticity, including stabilization of synaptic plasticity. As used herein,
"defect in synaptic
plasticity" or "aberrant synaptic plasticity" refers to abnormal synaptic
plasticity following
stimulation of that synapse. In some embodiments, a defect in synaptic
plasticity is a
decrease in LTP. In some embodiments, a defect in synaptic plasticity is an
increase in
LTD. In some embodiments, a defect in synaptic plasticity is erratic (e.g.,
fluctuating,
randomly increasing or decreasing) synaptic plasticity. In some instances,
measures of
synaptic plasticity are LTP and/or LTD (induced, for example, by theta-burst
stimulation,
high-frequency stimulation for LTP, low-frequency (e.g., e.g., 1 Hz)
stimulation for LTD)
and LTP and/or LTD after stabilization. In some embodiments, stabilization of
LTP and/or
LTD occurs in any region of the brain including the frontal cortex, the
hippocampus, the
prefrontal cortex, the amygdala or any combination thereof.
[00163] As used herein "stabilization of synaptic plasticity" refers to stable
LTP or LTD
following induction (e.g., by theta-burst stimulation, high-frequency
stimulation for LTP,
low-frequency (e.g., e.g., 1 Hz) stimulation for LTD).
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[00164] "Aberrant stabilization of synaptic transmission" (for example,
aberrant
stabilization of LTP or LTD), refers to failure to establish a stable baseline
of synaptic
transmission following an induction paradigm (e.g., by theta-burst
stimulation, high-
frequency stimulation for LTP, low-frequency (e.g., 1 Hz) stimulation for LTD)
or an
extended period of vulnerability to disruption by pharmacological or
electrophysiological
means
[00165] As used herein "synaptic transmission" or "baseline synaptic
transmission"
refers to the EPSP and/or IPSP amplitude and frequency, neuronal excitability
or population
spike thresholds of a normal individual (e.g., an individual not suffering
from a CNS
disorder) or that predicted for an animal model for a normal individual. As
used herein
"aberrant synaptic transmission" or "defective synaptic transmission" refers
to any
deviation in synaptic transmission compared to synaptic transmission of a
normal individual
or that predicted for an animal model for a normal individual. In some
embodiments, an
individual suffering from a CNS disorder has a defect in baseline synaptic
transmission that
is a decrease in baseline synaptic transmission compared to the baseline
synaptic
transmission in a normal individual or that predicted for an animal model for
a normal
individual. In some embodiments, an individual suffering from a CNS disorder
has a defect
in baseline synaptic transmission that is an increase in baseline synaptic
transmission
compared to the baseline synaptic transmission in a normal individual or that
predicted for
an animal model for a normal individual.
[00166] As used herein "sensorimotor gating" is assessed, for example, by
measuring
prepulse inhibition (PPI) and/or habituation of the human startle response. In
some
embodiments, a defect in sensorimotor gating is a deficit in sensorimotor
gating. In some
embodiments, a defect in sensorimotor gating is an enhancement of sensorimotor
gating.
[00167] As used herein, "normalization of aberrant synaptic plasticity" refers
to a change
in aberrant synaptic plasticity in an individual suffering from, suspected of
having, or pre-
disposed to a CNS disorder to a level of synaptic plasticity that is
substantially the same as
the synaptic plasticity of a normal individual or to that predicted from an
animal model for a
normal individual. As used herein, substantially the same means, for example,
about 90% to
about 110% of the measured synaptic plasticity in a normal individual or to
that predicted
from an animal model for a normal individual. In other embodiments,
substantially the same
means, for example, about 80% to about 120% of the measured synaptic
plasticity in a
normal individual or to that predicted from an animal model for a normal
individual. In yet
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other embodiments, substantially the same means, for example, about 70% to
about 130%
of the synaptic plasticity in a normal individual or to that predicted from an
animal model
for a normal individual. As used herein, "partial normalization of aberrant
synaptic
plasticity" refers to any change in aberrant synaptic plasticity in an
individual suffering
from, suspected of having, or pre-disposed to a CNS disorder that trends
towards synaptic
plasticity of a normal individual or to that predicted from an animal model
for a normal
individual. As used herein "partially normalized synaptic plasticity" or
"partially normal
synaptic plasticity" is, for example, about 25%, about 35%, about 45%,
about 55%,
about 65%, or about 75% of the synaptic plasticity of a normal individual or
to that
predicted from an animal model for a normal individual. In some embodiments,
normalization or partial normalization of aberrant synaptic plasticity in an
individual
suffering from, suspected of having, or pre-disposed to a CNS disorder is
lowering of
aberrant synaptic plasticity where the aberrant synaptic plasticity is higher
than the synaptic
plasticity of a normal individual or to that predicted from an animal model
for a normal
individual. In some embodiments, normalization or partial normalization of
aberrant
synaptic plasticity in an individual suffering from, suspected of having, or
pre-disposed to a
CNS disorder is an increase in aberrant synaptic plasticity where the aberrant
synaptic
plasticity is lower than the synaptic plasticity of a normal individual or to
that predicted
from an animal model for a normal individual. In some embodiments,
normalization or
partial normalization of synaptic plasticity in an individual suffering from,
suspected of
having, or pre-disposed to a CNS disorder is a change from an erratic (e.g.,
fluctuating,
randomly increasing or decreasing) synaptic plasticity to a normal (e.g.
stable) or partially
normal (e.g., less fluctuating) synaptic plasticity compared to the synaptic
plasticity of a
normal individual or to that predicted from an animal model for a normal
individual. In
some embodiments, normalization or partial normalization of synaptic
plasticity in an
individual suffering from, suspected of having, or pre-disposed to a CNS
disorder is a
change from a non-stabilizing synaptic plasticity to a normal (e.g., stable)
or partially
normal (e.g., partially stable) synaptic plasticity compared to the synaptic
plasticity of a
normal individual or to that predicted from an animal model for a normal
individual.
[00168] As used herein, "normalization of aberrant baseline synaptic
transmission" refers
to a change in aberrant baseline synaptic transmission in an individual
suffering from,
suspected of having, or pre-disposed to a CNS disorder to a level of baseline
synaptic
transmission that is substantially the same as the baseline synaptic
transmission of a normal
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individual or to that predicted from an animal model for a normal individual.
As used
herein, substantially the same means, for example, about 90% to about 110% of
the
measured baseline synaptic transmission in a normal individual or to that
predicted from an
animal model for a normal individual. In other embodiments, substantially the
same means,
for example, about 80% to about 120% of the measured baseline synaptic
transmission in a
normal individual or to that predicted from an animal model for a normal
individual. In yet
other embodiments, substantially the same means, for example, about 70% to
about 130%
of the measured baseline synaptic transmission in a normal individual or to
that predicted
from an animal model for a normal individual. As used herein, "partial
normalization of
aberrant baseline synaptic transmission" refers to any change in aberrant
baseline synaptic
transmission in an individual suffering from, suspected of having, or pre-
disposed to a CNS
disorder that trends towards baseline synaptic transmission of a normal
individual or to that
predicted from an animal model for a normal individual. As used herein
"partially
normalized baseline synaptic transmission" or "partially normal baseline
synaptic
transmission" is, for example, about 25%, about 35%, about 45%, about
55%,
about 65%, or about 75% of the measured baseline synaptic transmission of a
normal
individual or to that predicted from an animal model for a normal individual.
In some
embodiments, normalization or partial normalization of aberrant baseline
synaptic
transmission in an individual suffering from, suspected of having, or pre-
disposed to a CNS
disorder is lowering of aberrant baseline synaptic transmission where the
aberrant baseline
synaptic transmission is higher than the baseline synaptic transmission of a
normal
individual or to that predicted from an animal model for a normal individual.
In some
embodiments, normalization or partial normalization of aberrant baseline
synaptic
transmission in an individual suffering from, suspected of having, or pre-
disposed to a CNS
disorder is an increase in aberrant baseline synaptic transmission where the
aberrant
baseline synaptic transmission is lower than the baseline synaptic
transmission of a normal
individual or to that predicted from an animal model for a normal individual.
In some
embodiments, normalization or partial normalization of baseline synaptic
transmission in an
individual suffering from, suspected of having, or pre-disposed to a CNS
disorder is a
change from an erratic (e.g., fluctuating, randomly increasing or decreasing)
baseline
synaptic transmission to a normal (e.g. stable) or partially normal (e.g.,
less fluctuating)
baseline synaptic transmission compared to the baseline synaptic transmission
of a normal
individual or to that predicted from an animal model for a normal individual.
In some
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embodiments, normalization or partial normalization of aberrant baseline
synaptic
transmission in an individual suffering from, suspected of having, or pre-
disposed to a CNS
disorder is a change from a non-stabilizing baseline synaptic transmission to
a normal (e.g.,
stable) or partially normal (e.g., partially stable) baseline synaptic
transmission compared to
the baseline synaptic transmission of a normal individual or to that predicted
from an animal
model for a normal individual.
[00169] As used herein, "normalization of aberrant synaptic function" refers
to a change
in aberrant synaptic function in an individual suffering from, suspected of
having, or pre-
disposed to a CNS disorder to a level of synaptic function that is
substantially the same as
the synaptic function of a normal individual or to that predicted from an
animal model for a
normal individual. As used herein, substantially the same means, for example,
about 90% to
about 110% of the synaptic function in a normal individual or to that
predicted from an
animal model for a normal individual. In other embodiments, substantially the
same means,
for example, about 80% to about 120% of the synaptic function in a normal
individual or to
that predicted from an animal model for a normal individual. In yet other
embodiments,
substantially the same means, for example, about 70% to about 130% of the
synaptic
function in a normal individual or to that predicted from an animal model for
a normal
individual. As used herein, "partial normalization of aberrant synaptic
function" refers to
any change in aberrant synaptic function in an individual suffering from,
suspected of
having, or pre-disposed to a CNS disorder that trends towards synaptic
function of a normal
individual or to that predicted from an animal model for a normal individual.
As used herein
"partially normalized synaptic function" or "partially normal synaptic
function" is, for
example, about 25%, about 35%, about 45%, about 55%, about 65%, or
about
75% of the measured synaptic function of a normal individual or to that
predicted from an
animal model for a normal individual. In some embodiments, normalization or
partial
normalization of aberrant synaptic function in an individual suffering from,
suspected of
having, or pre-disposed to a CNS disorder is lowering of aberrant synaptic
function where
the aberrant synaptic function is higher than the synaptic function of a
normal individual or
to that predicted from an animal model for a normal individual. In some
embodiments,
normalization or partial normalization of aberrant synaptic function in an
individual
suffering from, suspected of having, or pre-disposed to a CNS disorder is an
increase in
aberrant synaptic function where the aberrant synaptic function is lower than
the synaptic
function of a normal individual or to that predicted from an animal model for
a normal
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individual. In some embodiments, normalization or partial normalization of
synaptic
function in an individual suffering from, suspected of having, or pre-disposed
to a CNS
disorder is a change from an erratic (e.g., fluctuating, randomly increasing
or decreasing)
synaptic function to a normal (e.g. stable) or partially normal (e.g., less
fluctuating) synaptic
function compared to the synaptic function of a normal individual or to that
predicted from
an animal model for a normal individual. In some embodiments, normalization or
partial
normalization of aberrant synaptic function in an individual suffering from,
suspected of
having, or pre-disposed to a CNS disorder is a change from a non-stabilizing
synaptic
function to a normal (e.g., stable) or partially normal (e.g., partially
stable) synaptic function
compared to the synaptic function of a normal individual or to that predicted
from an animal
model for a normal individual.
[00170] As used herein, "normalization of aberrant long term potentiation
(LTP)" refers
to a change in aberrant LTP in an individual suffering from, suspected of
having, or pre-
disposed to a CNS disorder to a level of LTP that is substantially the same as
the LTP of a
normal individual or to that predicted from an animal model for a normal
individual. As
used herein, substantially the same means, for example, about 90% to about
110% of the
LTP in a normal individual or to that predicted from an animal model for a
normal
individual. In other embodiments, substantially the same means, for example,
about 80% to
about 120% of the LTP in a normal individual or to that predicted from an
animal model for
a normal individual. In yet other embodiments, substantially the same means,
for example,
about 70% to about 130% of the LTP in a normal individual or to that predicted
from an
animal model for a normal individual. As used herein, "partial normalization
of aberrant
LTP" refers to any change in aberrant LTP in an individual suffering from,
suspected of
having, or pre-disposed to a CNS disorder that trends towards LTP of a normal
individual or
to that predicted from an animal model for a normal individual. As used herein
"partially
normalized LTP" or "partially normal LTP" is, for example, about 25%,
about 35%,
about 45%, about 55%, about 65%, or about 75% of the measured LTP of a
normal
individual or to that predicted from an animal model for a normal individual.
In some
embodiments, normalization or partial normalization of aberrant LTP in an
individual
suffering from, suspected of having, or pre-disposed to a CNS disorder is
lowering of
aberrant LTP where the aberrant LTP is higher than the LTP of a normal
individual or to
that predicted from an animal model for a normal individual. In some
embodiments,
normalization or partial normalization of aberrant LTP in an individual
suffering from,
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suspected of having, or pre-disposed to a CNS disorder is an increase in
aberrant LTP where
the aberrant LTP is lower than the LTP of a normal individual or to that
predicted from an
animal model for a normal individual. In some embodiments, normalization or
partial
normalization of LTP in an individual suffering from, suspected of having, or
pre-disposed
to a CNS disorder is a change from an erratic (e.g., fluctuating, randomly
increasing or
decreasing) LTP to a normal (e.g. stable) or partially normal (e.g., less
fluctuating) LTP
compared to the LTP of a normal individual or to that predicted from an animal
model for a
normal individual. In some embodiments, normalization or partial normalization
of aberrant
LTP in an individual suffering from, suspected of having, or pre-disposed to a
CNS disorder
is a change from a non-stabilizing LTP to a normal (e.g., stable) or partially
normal (e.g.,
partially stable) LTP compared to the LTP of a normal individual or to that
predicted from
an animal model for a normal individual.
[00171] As used herein, "normalization of aberrant long term depression (LTD)"
refers
to a change in aberrant LTD in an individual suffering from, suspected of
having, or pre-
disposed to a CNS disorder to a level of LTD that is substantially the same as
the LTD of a
normal individual or to that predicted from an animal model for a normal
individual. As
used herein, substantially the same means, for example, about 90% to about
110% of the
LTD in a normal individual or to that predicted from an animal model for a
normal
individual. In other embodiments, substantially the same means, for example,
about 80% to
about 120% of the LTD in a normal individual or to that predicted from an
animal model for
a normal individual. In yet other embodiments, substantially the same means,
for example,
about 70% to about 130% of the LTD in a normal individual or to that predicted
from an
animal model for a normal individual. As used herein, "partial normalization
of aberrant
LTD" refers to any change in aberrant LTD in an individual suffering from,
suspected of
having, or pre-disposed to a CNS disorder that trends towards LTD of a normal
individual
or to that predicted from an animal model for a normal individual. As used
herein "partially
normalized LTD" or "partially normal LTD" is, for example, about 25%,
about 35%,
about 45%, about 55%, about 65%, or about 75% of the measured LTD of a
normal
individual or to that predicted from an animal model for a normal individual.
In some
embodiments, normalization or partial normalization of aberrant LTD in an
individual
suffering from, suspected of having, or pre-disposed to a CNS disorder is
lowering of
aberrant LTD where the aberrant LTD is higher than the LTD of a normal
individual or to
that predicted from an animal model for a normal individual. In some
embodiments,
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normalization or partial normalization of aberrant LTD in an individual
suffering from,
suspected of having, or pre-disposed to a CNS disorder is an increase in
aberrant LTD
where the aberrant LTD is lower than the LTD of a normal individual or to that
predicted
from an animal model for a normal individual. In some embodiments,
normalization or
partial normalization of LTD in an individual suffering from, suspected of
having, or pre-
disposed to a CNS disorder is a change from an erratic (e.g., fluctuating,
randomly
increasing or decreasing) LTD to a normal (e.g. stable) or partially normal
(e.g., less
fluctuating) LTD compared to the LTD of a normal individual or to that
predicted from an
animal model for a normal individual. In some embodiments, normalization or
partial
normalization of aberrant LTD in an individual suffering from, suspected of
having, or pre-
disposed to a CNS disorder is a change from a non-stabilizing LTD to a normal
(e.g., stable)
or partially normal (e.g., partially stable) LTD compared to the LTD of a
normal individual
or to that predicted from an animal model for a normal individual.
[00172] As used herein, "normalization of aberrant sensorimotor gating" refers
to a
change in aberrant sensorimotor gating in an individual suffering from,
suspected of having,
or pre-disposed to a CNS disorder to a level of sensorimotor gating that is
substantially the
same as the sensorimotor gating of a normal individual or to that predicted
from an animal
model for a normal individual. As used herein, substantially the same means,
for example,
about 90% to about 110% of the sensorimotor gating in a normal individual or
to that
predicted from an animal model for a normal individual. In other embodiments,
substantially the same means, for example, about 80% to about 120% of the
sensorimotor
gating in a normal individual or to that predicted from an animal model for a
normal
individual. In yet other embodiments, substantially the same means, for
example, about
70% to about 130% of the sensorimotor gating in a normal individual or to that
predicted
from an animal model for a normal individual. As used herein, "partial
normalization of
aberrant sensorimotor gating" refers to any change in aberrant sensorimotor
gating in an
individual suffering from, suspected of having, or pre-disposed to a CNS
disorder that
trends towards sensorimotor gating of a normal individual or to that predicted
from an
animal model for a normal individual. As used herein "partially normalized
sensorimotor
gating" or "partially normal sensorimotor gating" is, for example, about
25%, about
35%, about 45%, about 55%, about 65%, or about 75% of the measured
sensorimotor gating of a normal individual or to that predicted from an animal
model for a
normal individual. In some embodiments, normalization or partial normalization
of aberrant
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sensorimotor gating in an individual suffering from, suspected of having, or
pre-disposed to
a CNS disorder is lowering of aberrant sensorimotor gating where the aberrant
sensorimotor
gating is higher than the sensorimotor gating of a normal individual or to
that predicted
from an animal model for a normal individual. In some embodiments,
normalization or
partial normalization of aberrant sensorimotor gating in an individual
suffering from,
suspected of having, or pre-disposed to a CNS disorder is an increase in
aberrant
sensorimotor gating where the aberrant sensorimotor gating is lower than the
sensorimotor
gating of a normal individual or to that predicted from an animal model for a
normal
individual. In some embodiments, normalization or partial normalization of
sensorimotor
gating in an individual suffering from, suspected of having, or pre-disposed
to a CNS
disorder is a change from an erratic (e.g., fluctuating, randomly increasing
or decreasing)
sensorimotor gating to a normal (e.g. stable) or partially normal (e.g., less
fluctuating)
sensorimotor gating compared to the sensorimotor gating of a normal individual
or to that
predicted from an animal model for a normal individual. In some embodiments,
normalization or partial normalization of aberrant sensorimotor gating in an
individual
suffering from, suspected of having, or pre-disposed to a CNS disorder is a
change from a
non-stabilizing sensorimotor gating to a normal (e.g., stable) or partially
normal (e.g.,
partially stable) sensorimotor gating compared to the sensorimotor gating of a
normal
individual or to that predicted from an animal model for a normal individual.
[00173] As used herein, "expression" of a nucleic acid sequence refers to one
or more of
the following events: (1) production of an RNA template from a DNA sequence
(e.g., by
transcription); (2) processing of an RNA transcript (e.g., by splicing,
editing, 5' cap
formation, and/or 3' end formation); (3) translation of an RNA into a
polypeptide or protein;
(4) post-translational modification of a polypeptide or protein.
[00174] As used herein the term "PAK polypeptide" or "PAK protein" or "PAK"
refers
to a protein that belongs in the family of p21-activated serine/threonine
protein kinases.
These include mammalian isoforms of PAK, e.g., the Group I PAK proteins
(sometimes
referred to as Group A PAK proteins), including PAK 1, PAK2, PAK3, as well as
the Group
II PAK proteins (sometimes referred to as Group B PAK proteins), including
PAK4, PAKS,
and/or PAK6 Also included as PAK polypeptides or PAK proteins are lower
eukaryotic
isoforms, such as the yeast Ste20 (Leberter et al., 1992, EMBO J., 11:4805;
incorporated
herein by reference) and/or the Dictyostelium single-headed myosin I heavy
chain kinases
(Wu et al., 1996, J. Biol. Chem., 271:31787; incorporated herein by
reference).
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Representative examples of PAK amino acid sequences include, but are not
limited to,
human PAK1 (GenBank Accession Number AAA65441), human PAK2 (GenBank
Accession Number AAA65442), human PAK3 (GenBank Accession Number AAC36097),
human PAK 4 (GenBank Accession Numbers NP005875 and CAA09820), human PAK5
(GenBank Accession Numbers CAC18720 and BAA94194), human PAK6 (GenBank
Accession Numbers NP064553 and AAF82800), human PAK7 (GenBank Accession
Number Q9P286), C. elegans PAK (GenBank Accession Number BAA11844), D.
melanogaster PAK (GenBank Accession Number AAC47094), and rat PAK1 (GenBank
Accession Number AAB95646). In some embodiments, a PAK polypeptide comprises
an
amino acid sequence that is at least 70% to 100% identical, e.g., at least
75%, 80%, 85%,
86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent
from
about 70% to about 100% identical to sequences of GenBank Accession Numbers
AAA65441, AAA65442, AAC36097, NP 005875, CAA09820, CAC18720, BAA94194,
NP064553, AAF82800, Q9P286, BAA11844, AAC47094, and/or AAB95646. In some
embodiments, a Group I PAK polypeptide comprises an amino acid sequence that
is at least
70% to 100% identical, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%,
92%,
94%, 95%, 96%, 97%, 98%, or any other percent from about 70% to about 100%
identical
to sequences of GenBank Accession Numbers AAA65441, AAA65442, and/or AAC36097.
[00175] Representative examples of PAK genes encoding PAK proteins include,
but are
not limited to, human PAK1 (GenBank Accession Number U24152), human PAK2
(GenBank Accession Number U24153), human PAK3 (GenBank Accession Number
AF068864), human PAK4 (GenBank Accession Number AJ011855), human PAK5
(GenBank Accession Number AB040812), and human PAK6 (GenBank Accession Number
AF276893). In some embodiments, a PAK gene comprises a nucleotide sequence
that is at
least 70% to 100% identical, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 90%,
91%,
92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 70% to about
100%
identical to sequences of GenBank Accession Numbers U24152, U24153, AF068864,
AJ011855, AB040812, and/or AF276893. In some embodiments, a Group I PAK gene
comprises a nucleotide sequence that is at least 70% to 100% identical, e.g.,
at least 75%,
80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other
percent from about 70% to about 100% identical to sequences of GenBank
Accession
Numbers U24152, U24153, and/or AF068864.
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[00176] To determine the percent homology of two amino acid sequences or of
two
nucleic acids, the sequences are aligned for optimal comparison purposes
(e.g., gaps can be
introduced in the sequence of a first amino acid or nucleic acid sequence for
optimal
alignment with a second amino or nucleic acid sequence). The amino acid
residues or
nucleotides at corresponding amino acid positions or nucleotide positions are
then
compared. When a position in the first sequence is occupied by the same amino
acid residue
or nucleotide as the corresponding position in the second sequence, then the
molecules are
identical at that position. The percent homology between the two sequences is
a function of
the number of identical positions shared by the sequences (i.e., % identity =
# of identical
positions/total # of positions (e.g., overlapping positions) x 100). In one
embodiment the
two sequences are the same length.
[00177] To determine percent homology between two sequences, the algorithm of
Karlin
and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in
Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877 is used. Such an
algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J.
Mol.
Biol. 215:403-410. BLAST nucleotide searches are performed with the NBLAST
program,
score=100, wordlength=12 to obtain nucleotide sequences homologous to a
nucleic acid
molecules described or disclose herein. BLAST protein searches are performed
with the
XBLAST program, score=50, wordlength=3. To obtain gapped alignments for
comparison
purposes, Gapped BLAST is utilized as described in Altschul et al. (1997)
Nucleic Acids
Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See
the
website of the National Center for Biotechnology Information for further
details (on the
world wide web at ncbi.nlm.nih.gov). Proteins suitable for use in the methods
described
herein also includes proteins having between 1 to 15 amino acid changes, e.g.,
1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, deletions, or
additions,
compared to the amino acid sequence of any protein PAK inhibitor described
herein. In
other embodiments, the altered amino acid sequence is at least 75% identical,
e.g., 77%,
80%, 82%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to the
amino
acid sequence of any protein PAK inhibitor described herein. Such sequence-
variant
proteins are suitable for the methods described herein as long as the altered
amino acid
sequence retains sufficient biological activity to be functional in the
compositions and
methods described herein. Where amino acid substitutions are made, the
substitutions
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should be conservative amino acid substitutions. Among the common amino acids,
for
example, a "conservative amino acid substitution" is illustrated by a
substitution among
amino acids within each of the following groups: (1) glycine, alanine, valine,
leucine, and
isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and
threonine, (4)
aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine,
arginine and histidine.
The BLOSUM62 table is an amino acid substitution matrix derived from about
2,000 local
multiple alignments of protein sequence segments, representing highly
conserved regions of
more than 500 groups of related proteins (Henikoff et al (1992), Proc. Natl
Acad. Sci. USA,
89:10915-10919). Accordingly, the BLOSUM62 substitution frequencies are used
to define
conservative amino acid substitutions that may be introduced into the amino
acid sequences
described or described herein. Although it is possible to design amino acid
substitutions
based solely upon chemical properties (as discussed above), the language
"conservative
amino acid substitution" preferably refers to a substitution represented by a
BLOSUM62
value of greater than -1. For example, an amino acid substitution is
conservative if the
substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. According
to this
system, preferred conservative amino acid substitutions are characterized by a
BLOSUM62
value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino
acid
substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or
3).
[00178] As used herein, the term "PAK activity," unless otherwise specified,
includes,
but is not limited to, at least one of PAK protein-protein interactions, PAK
phosphotransferase activity (intermolecular or intermolecular), translocation,
etc of one or
more PAK isoforms.
[00179] As used herein, a "PAK inhibitor" refers to any molecule, compound, or
composition that directly or indirectly decreases the PAK activity. In some
embodiments,
PAK inhibitors inhibit, decrease, and/or abolish the level of a PAK mRNA
and/or protein or
the half-life of PAK mRNA and/or protein, such inhibitors are referred to as
"clearance
agents". In some embodiments, a PAK inhibitor is a PAK antagonist that
inhibits,
decreases, and/or abolishes an activity of PAK. In some embodiments, a PAK
inhibitor also
disrupts, inhibits, or abolishes the interaction between PAK and its natural
binding partners
(e.g., a substrate for a PAK kinase, a Rae protein, a cdc42 protein, LIM
kinase) or a protein
that is a binding partner of PAK in a pathological condition, as measured
using standard
methods. In some embodiments, the PAK inhibitor is a Group I PAK inhibitor
that inhibits,
for example, one or more Group I PAK polypeptides, for example, PAK1, PAK2,
and/or
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PAK3. In some embodiments, the PAK inhibitor is a PAK1 inhibitor. In some
embodiments, the PAK inhibitor is a PAK2 inhibitor. In some embodiments, the
PAK
inhibitor is a PAK3 inhibitor. In some embodiments, the PAK inhibitor is a
mixed
PAK1/PAK3 inhibitor. In some embodiments, the PAK inhibitor inhibits all three
Group I
PAK isoforms (PAK1, PAK2 and PAK3) with equal or similar potency. In some
embodiments, the PAK inhibitor is a Group II PAK inhibitor that inhibits one
or more
Group II PAK polypeptides, for example PAK4, PAK5, and/or PAK6. In some
embodiments, the PAK inhibitor is a PAK4 inhibitor. In some embodiments, the
PAK
inhibitor is a PAK5 inhibitor. In some embodiments, the PAK inhibitor is a
PAK6 inhibitor.
In some embodiments, the PAK inhibitor is a PAK7 inhibitor. As used herein, a
PAK5
polypeptide is substantially homologous to a PAK7 polypeptide.
[00180] In some embodiments, PAK inhibitors reduce, abolish, and/or remove the
binding between PAK and at least one of its natural binding partners (e.g.,
Cdc42 or Rac).
In some instances, binding between PAK and at least one of its natural binding
partners is
stronger in the absence of a PAK inhibitor (by e.g., 90%, 80%, 70%, 60%, 50%,
40%, 30%
or 20%) than in the presence of a PAK inhibitor. In some embodiments, PAK
inhibitors
prevent, reduce, or abolish binding between PAK and a protein that abnormally
accumulates
or aggregates in cells or tissue in a disease state. In some instances,
binding between PAK
and at least one of the proteins that aggregates or accumulates in a cell or
tissue is stronger
in the absence of a PAK inhibitor (by e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30%
or 20%)
than in the presence of an inhibitor.
[00181] An "individual" or an "individual," as used herein, is a mammal. In
some
embodiments, an individual is an animal, for example, a rat, a mouse, a dog or
a monkey. In
some embodiments, an individual is a human patient. In some embodiments an
"individual"
or an "individual" is a human. In some embodiments, an individual suffers from
a CNS
disorder or is suspected to be suffering from a CNS disorder or is pre-
disposed to a CNS
disorder.
[00182] In some embodiments, a pharmacological composition comprising a PAK
inhibitor is "administered peripherally" or "peripherally administered." As
used herein,
these terms refer to any form of administration of an agent, e.g., a
therapeutic agent, to an
individual that is not direct administration to the CNS, i.e., that brings the
agent in contact
with the non-brain side of the blood-brain barrier. "Peripheral
administration," as used
herein, includes intravenous, intra-arterial, subcutaneous, intramuscular,
intraperitoneal,
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transdermal, by inhalation, transbuccal, intranasal, rectal, oral, parenteral,
sublingual, or
trans-nasal. In some embodiments, a PAK inhibitor is administered by an
intracerebral
route.
[00183] The terms "polypeptide," and "protein" are used interchangeably herein
to refer
to a polymer of amino acid residues. That is, a description directed to a
polypeptide applies
equally to a description of a protein, and vice versa. The terms apply to
naturally occurring
amino acid polymers as well as amino acid polymers in which one or more amino
acid
residues is a non-naturally occurring amino acid, e.g., an amino acid analog.
As used herein,
the terms encompass amino acid chains of any length, including full length
proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent peptide
bonds.
[00184] The term "amino acid" refers to naturally occurring and non-naturally
occurring
amino acids, as well as amino acid analogs and amino acid mimetics that
function in a
manner similar to the naturally occurring amino acids. Naturally encoded amino
acids are
the 20 common amino acids (alanine, arginine, asparagine, aspartic acid,
cysteine,
glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine,
methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine)
and pyrolysine
and selenocysteine. Amino acid analogs refers to compounds that have the same
basic
chemical structure as a naturally occurring amino acid, i.e., an a carbon that
is bound to a
hydrogen, a carboxyl group, an amino group, and an R group, such as,
homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs
have
modified R groups (such as, norleucine) or modified peptide backbones, but
retain the same
basic chemical structure as a naturally occurring amino acid.
[00185] Amino acids may be referred to herein by either their commonly known
three
letter symbols or by the one-letter symbols recommended by the IUPAC-IUB
Biochemical
Nomenclature Commission. Nucleotides, likewise, may be referred to by their
commonly
accepted single-letter codes.
[00186] The term "nucleic acid" refers to deoxyribonucleotides,
deoxyribonucleosides,
ribonucleosides, or ribonucleotides and polymers thereof in either single- or
double-
stranded form. Unless specifically limited, the term encompasses nucleic acids
containing
known analogues of natural nucleotides which have similar binding properties
as the
reference nucleic acid and are metabolized in a manner similar to naturally
occurring
nucleotides. Unless specifically limited otherwise, the term also refers to
oligonucleotide
analogs including PNA (peptidonucleic acid), analogs of DNA used in antisense
technology
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(phosphorothioates, phosphoroamidates, and the like). Unless otherwise
indicated, a
particular nucleic acid sequence also implicitly encompasses conservatively
modified
variants thereof (including but not limited to, degenerate codon
substitutions) and
complementary sequences as well as the sequence explicitly indicated.
Specifically,
degenerate codon substitutions may be achieved by generating sequences in
which the third
position of one or more selected (or all) codons is substituted with mixed-
base and/or
deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);
Ohtsuka et al., J.
Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992); Rossolini et al.,
Mol. Cell.
Probes 8:91-98 (1994)).
[00187] The terms "isolated" and "purified" refer to a material that is
substantially or
essentially removed from or concentrated in its natural environment. For
example, an
isolated nucleic acid is one that is separated from the nucleic acids that
normally flank it or
other nucleic acids or components (proteins, lipids, etc.) in a sample. In
another example, a
polypeptide is purified if it is substantially removed from or concentrated in
its natural
environment. Methods for purification and isolation of nucleic acids and
proteins are
documented methodologies.
[00188] The term "antibody" describes an immunoglobulin whether natural or
partly or
wholly synthetically produced. The term also covers any polypeptide or protein
having a
binding domain which is, or is homologous to, an antigen-binding domain. CDR
grafted
antibodies are also contemplated by this term.
[00189] The term antibody as used herein will also be understood to mean one
or more
fragments of an antibody that retain the ability to specifically bind to an
antigen, (see
generally, Holliger et al., Nature Biotech. 23 (9) 1126-1129 (2005)). Non-
limiting examples
of such antibodies include (i) a Fab fragment, a monovalent fragment
consisting of the VL,
VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment
comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a Fd
fragment consisting of
the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains
of a
single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544 546),
which consists of a VH domain; and (vi) an isolated complementarity
determining region
(CDR). Furthermore, although the two domains of the Fv fragment, VL and VH,
are coded
for by separate genes, they are optionally joined, using recombinant methods,
by a synthetic
linker that enables them to be made as a single protein chain in which the VL
and VH
regions pair to form monovalent molecules (known as single chain Fv (scFv);
see e.g., Bird
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et al. (1988) Science 242:423 426; and Huston et al. (1988) Proc. Natl. Acad.
Sci. USA
85:5879 5883; and Osbourn et al. (1998) Nat. Biotechnol. 16:778). Such single
chain
antibodies are also intended to be encompassed within the term antibody. Any
VH and VL
sequences of specific scFv is optionally linked to human immunoglobulin
constant region
cDNA or genomic sequences, in order to generate expression vectors encoding
complete
IgG molecules or other isotypes. VH and VL are also optionally used in the
generation of
Fab, Fv or other fragments of immunoglobulins using either protein chemistry
or
recombinant DNA technology. Other forms of single chain antibodies, such as
diabodies are
also encompassed.
l0 [00190] "F(ab')2" and "Fab"` moieties are optionally produced by treating
immunoglobulin (monoclonal antibody) with a protease such as pepsin and
papain, and
includes an antibody fragment generated by digesting immunoglobulin near the
disulfide
bonds existing between the hinge regions in each of the two H chains. For
example, papain
cleaves IgG upstream of the disulfide bonds existing between the hinge regions
in each of
the two H chains to generate two homologous antibody fragments in which an L
chain
composed of VL (L chain variable region) and CL (L chain constant region), and
an H chain
fragment composed of VH (H chain variable region) and CHyl (yl region in the
constant
region of H chain) are connected at their C terminal regions through a
disulfide bond. Each
of these two homologous antibody fragments is called Fab'. Pepsin also cleaves
IgG
downstream of the disulfide bonds existing between the hinge regions in each
of the two H
chains to generate an antibody fragment slightly larger than the fragment in
which the two
above-mentioned Fab' are connected at the hinge region. This antibody fragment
is called
F(ab')2.
[00191] The Fab fragment also contains the constant domain of the light chain
and the
first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab
fragments
by the addition of a few residues at the carboxyl terminus of the heavy chain
CH1 domain
including one or more cysteine(s) from the antibody hinge region. Fab'-SH is
the
designation herein for Fab' in which the cysteine residue(s) of the constant
domains bear a
free thiol group. F(ab')2 antibody fragments originally were produced as pairs
of Fab'
fragments which have hinge cysteines between them. Other chemical couplings of
antibody
fragments are documented.
[00192] "Fv" is the minimum antibody fragment which contains a complete
antigen-
recognition and antigen-binding site. This region consists of a dimer of one
heavy chain and
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one light chain variable domain in tight, non-covalent association. It is in
this configuration
that the three hypervariable regions of each variable domain interact to
define an antigen-
binding site on the surface of the VH-VL dimer. Collectively, the six
hypervariable regions
confer antigen-binding specificity to the antibody. However, even a single
variable domain
(or half of an Fv comprising only three hypervariable regions specific for an
antigen) has
the ability to recognize and bind antigen, although at a lower affinity than
the entire binding
site.
[00193] "Single-chain Fv" or "sFv" antibody fragments comprise a VH, a VL, or
both a
VH and VL domain of an antibody, wherein both domains are present in a single
polypeptide chain. In some embodiments, the Fv polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the sFv to form
the
desired structure for antigen binding. For a review of sFv see, e.g.,
Pluckthun in The
Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.
Springer-
Verlag, New York, pp. 269 315 (1994).
[00194] A "chimeric" antibody includes an antibody derived from a combination
of
different mammals. The mammal is, for example, a rabbit, a mouse, a rat, a
goat, or a
human. The combination of different mammals includes combinations of fragments
from
human and mouse sources.
[00195] In some embodiments, an antibody described or described herein is a
monoclonal antibody (MAb), typically a chimeric human-mouse antibody derived
by
humanization of a mouse monoclonal antibody. Such antibodies are obtained
from, e.g.,
transgenic mice that have been "engineered" to produce specific human
antibodies in
response to antigenic challenge. In this technique, elements of the human
heavy and light
chain locus are introduced into strains of mice derived from embryonic stem
cell lines that
contain targeted disruptions of the endogenous heavy chain and light chain
loci. In some
embodiments, the transgenic mice synthesize human antibodies specific for
human antigens,
and the mice are used to produce human antibody-secreting hybridomas.
[00196] The term "optionally substituted" or "substituted" means that the
referenced
group substituted with one or more additional group(s). In certain
embodiments, the one or
more additional group(s) are individually and independently selected from
amide, ester,
alkyl, cycloalkyl, heteroalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy,
alkoxy, aryloxy,
alkylthio, arylthio, alkylsulfoxide, arylsulfoxide, ester, alkylsulfone,
arylsulfone, cyano,
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halogen, alkoyl, alkoyloxo, isocyanato, thiocyanato, isothiocyanato, nitro,
haloalkyl,
haloalkoxy, fluoroalkyl, amino, alkyl-amino, dialkyl-amino, amido.
[00197] An "alkyl" group refers to an aliphatic hydrocarbon group. Reference
to an alkyl
group includes "saturated alkyl" and/or "unsaturated alkyl". The alkyl group,
whether
saturated or unsaturated, includes branched, straight chain, or cyclic groups.
By way of
example only, alkyl includes methyl, ethyl, propyl, iso-propyl, n-butyl, iso-
butyl, sec-butyl,
t-butyl, pentyl, iso-pentyl, neo-pentyl, and hexyl. In some embodiments, alkyl
groups
include, but are in no way limited to, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl,
tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl,
cyclobutyl,
cyclopentyl, cyclohexyl, and the like. A "lower alkyl" is a CI-C6 alkyl. A
"heteroalkyl"
group substitutes any one of the carbons of the alkyl group with a heteroatom
having the
appropriate number of hydrogen atoms attached (e.g., a CH2 group to an NH
group or an 0
group).
[00198] An "alkoxy" group refers to a (alkyl)O- group, where alkyl is as
defined herein.
[00199] The term "alkylamine" refers to the -N(alkyl)XHy group, wherein alkyl
is as
defined herein and x and y are selected from the group x=1, y=1 and x=2, y=0.
When x=2,
the alkyl groups, taken together with the nitrogen to which they are attached,
optionally
form a cyclic ring system.
[00200] An "amide" is a chemical moiety with formula C(O)NHR or NHC(O)R, where
R
is selected from alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring
carbon) and
heteroalicyclic (bonded through a ring carbon).
[00201] The term "ester" refers to a chemical moiety with formula -C(=O)OR,
where R
is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl
and
heteroalicyclic.
[00202] As used herein, the term "aryl" refers to an aromatic ring wherein
each of the
atoms forming the ring is a carbon atom. Aryl rings described herein include
rings having
five, six, seven, eight, nine, or more than nine carbon atoms. Aryl groups are
optionally
substituted. Examples of aryl groups include, but are not limited to phenyl,
and
naphthalenyl.
[00203] The term "cycloalkyl" refers to a monocyclic or polycyclic non-
aromatic radical,
wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon
atom. In various
embodiments, cycloalkyls are saturated, or partially unsaturated. In some
embodiments,
cycloalkyls are fused with an aromatic ring. Cycloalkyl groups include groups
having from
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3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include, but
are not limited to,
the following moieties:
z~6
> E (Do C 101-1
and the like. Monocyclic cycloalkyls include, but are not limited to,
cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Dicylclic
cycloalkyls
include, but are not limited to tetrahydronaphthyl, indanyl,
tetrahydropentalene or the like.
Polycyclic cycloalkyls include adamantane, norbornane or the like. The term
cycloalkyl
includes "unsaturated nonaromatic carbocyclyl" or "nonaromatic unsaturated
carbocyclyl"
groups both of which refer to a nonaromatic carbocycle, as defined herein,
that contains at
least one carbon carbon double bond or one carbon carbon triple bond.
[00204] The term "heterocyclo" refers to heteroaromatic and heteroalicyclic
groups
containing one to four ring heteroatoms each selected from 0, S and N. In
certain instances,
each heterocyclic group has from 4 to 10 atoms in its ring system, and with
the proviso that
the ring of said group does not contain two adjacent 0 or S atoms. Non-
aromatic
heterocyclic groups include groups having 3 atoms in their ring system, but
aromatic
heterocyclic groups must have at least 5 atoms in their ring system. The
heterocyclic groups
include benzo-fused ring systems. An example of a 3-membered heterocyclic
group is
aziridinyl (derived from aziridine). An example of a 4-membered heterocyclic
group is
azetidinyl (derived from azetidine). An example of a 5-membered heterocyclic
group is
thiazolyl. An example of a 6-membered heterocyclic group is pyridyl, and an
example of a
10-membered heterocyclic group is quinolinyl. Examples of non-aromatic
heterocyclic
groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,
tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino,
morpholino,
thiomorpholino, thioxanyl, piperazinyl, aziridinyl, azetidinyl, oxetanyl,
thietanyl,
homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl,
1,2,3,6-
tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-
pyranyl,
dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl,
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dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-
azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl and
quinolizinyl.
Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl,
pyrimidinyl, pyrazolyl,
triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,
oxazolyl, isothiazolyl,
pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,
cinnolinyl,
indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl,
pteridinyl, purinyl,
oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl,
benzothiazolyl,
benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.
[00205] The terms "heteroaryl" or, alternatively, "heteroaromatic" refers to
an aryl group
that includes one or more ring heteroatoms selected from nitrogen, oxygen and
sulfur. An
N-containing "heteroaromatic" or "heteroaryl" moiety refers to an aromatic
group in which
at least one of the skeletal atoms of the ring is a nitrogen atom. In certain
embodiments,
heteroaryl groups are monocyclic or polycyclic. Examples of monocyclic
heteroaryl groups
include and are not limited to:
H H H
\N/ \O/ \S/ \N N
/N _N
pyrrole furan thiophene pyrazole imidazole
(pyrrolyl) (furanyl) (thiophenyl) (pyrazolyl (imidazolyl)
H
CO/N CON CSN C
N CN
isoxazole oxazole isothiazole thiazolyl 1,2,3-triazole
(isoxazolyl) (oxazolyl (isothiazolyl) (thiazolyl) (1,2,3-triazolyl)
H 01 \\ N " 01 ,N \\ N\-/N
N-N N N ~/
1,3,4-triazole 1 -oxa-2,3-diazole 1-oxa-2,4-diazole 1-oxa-2,5-diazole
(1,3,4-triazolyl) (1-oxa-2,3-diazolyl) (1-oxa-2,4-diazolyl) (1-oxa-2,5-
diazolyl)
0 S, S, IS,
,N ,i Nv
N-N N N
1-oxa-3,4-diazole 1-thia-2,3-diazole 1-thia-2,4-diazole 1 -thia-2,5-diazole
(1-oxa-3, 4-diazolyl) (1-thia-2,3-diazolyl) (1-thia-2,4-diazolyl) (1-thia-2,5-
diazolyl)
H
NU-
N-N NN N
I S~ ~N,N N
N-N
1 -thia-3,4-diazole tetrazole pyridine pyridazine pyrimidine
(1-thia-3,4-diazolyl (tetrazolyl) (pyridinyl) (pyridazinyl) (pyrimidinyl)
CNJ
N N
pyrazine 1 ,3,5-triazine
(pyrazinyl) (triazinyl)
[00206] Examples of bicyclic heteroaryl groups include and are not limited to:
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N
S N/ N/ N
H H H
benzofuran benzothiophene indole benzimidazole indazole
(benzofuranyl) (benzothiaphenyl) (indolyl) (benzimidazolyl) (indazolyl)
,n\
NN N cccc?
benzotriazole pyrrolo[2,3-b]pyridine pyrrolo[2,3-c]pyridine pyrrolo[3,2-
c]pyridine
(benzotriazolyl) (pyrrolo[2,3-b]pyridinyl) (pyrrolo[2,3-c]pyridinyl)
(pyrrolo[3,2-c]pyridinyl)
H
N N~ N/ N, I N N
H N H H N
pyrrolo[3,2-b]pyridine imidazo[4,5-b]pyridine imidazo[4,5-c]pyridine
pyrazolo[4,3-d]pyridine
(pyrrolo[3,2-b]pyridinyl) (imidazo[4,5-b]pyridinyl) (imidazo[4,5-c]pyridinyl)
(pyrazolo[4,3-d]pyridinyl)
H H H
N, N N N, QNH
N ~N H ~N N pyrazolo[4,3-d]pyridine pyrazolo[3,4-c]pyridine pyrazolo[3,4-
b]pyridine isoindole
(pyrazolo[4,3-d]pyridinyl) (pyrazolo[3,4-c]pyridinyl) (pyrazolo[3,4-
b]pyridinyl) (isoindolyl)
N N~ / N> N Nom'/ CN-: N
H H
indazole purine indolizine imidazo[1,2-a]pyridine imidazo[1,5-a]pyridine
(indazolyl) (purinyl) (indolininyl) (imidazo[1,2-a]pyridinyl) (imidazo[1,5-
a]pyridinyl)
JN
\ N-N NN NN S N
pyrazolo[1,5-a]pyridine pyrrolo[1,2-b]pyridazine imidazo[1,2-c]pyrimidine
thienopyrimidine
(pyrazolo[1,5-a]pyridinyl) (pyrrolo[1,2-b]pyridazinyl) (imidazo[1,2-
c]pyrimidinyl) (thienopyrimidinyl)
<\,S- , rl,
N
thienopyrimidine
(thienopyrimidinyl)
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000CNOCNQfl
N N quinoline isoquinoline cinnoline quinazoline
(quinolinyl) (isoquinolinyl) (cinnolinyl) (azaquinazoline)
N~
iN \ N N
N N/ I / I
N
quinoxaline phthalazine 1,6-naphthyridine 1,7-naphthyridine
(quinoxalinyl) (phthalazinyl) (1,6-naphthyridinyl) (1,7-naphthyridinyl)
N N N N N~ I\ / I\
\ iN iN
1,8-naphthyridine 1,5-naphthyridine 2,6-naphthyridine 2,7-naphthyridine
(1,8-naphthyridinyl) (1,5-naphthyridinyl) (2,6-naphthyridinyl) (2,7-
naphthyridinyl)
N N~ N C1,1N
NNI J
N
pyrido[3,2-d]pyrimidine pyrido[4,3-d]pyrimidine pyrido[3,4-d]pyrimidine
(pyrido[3,2-d]pyrimidinyl) (pyrido[4,3-d]pyrimidinyl) (pyrido[3,4-
d]pyrimidinyl)
iN N N
N INN N \ I J
N
pyrido[2,3-d]pyrimidine pyrido[2,3-b]pyrazine pyrido[3,4-b]pyrazine
(pyrido[2,3-d]pyrimidinyl) (pyrido[2,3-b]pyrazinyl) (pyrido[3,4-b]pyrazinyl)
NN (I) V pyrimido[5,4-d]pyrimidine pyrazino[2,3-b]pyrazine pyrimido[4,5-
d]pyrimidine
(pyrido[5,4-d]pyrimidinyl) (pyrazino[2,3-b]pyrazinyl) (pyrido[4,5-
d]pyrimidinyl) or the like.
[00207] A "heteroalicyclic" group or "heterocyclo" group or "heterocycloalkyl"
group or
"heterocyclyl" group refers to a cycloalkyl group, wherein at least one
skeletal ring atom is
a heteroatom selected from nitrogen, oxygen and sulfur. In some embodiments,
the radicals
are fused with an aryl or heteroaryl. Example of saturated heterocyloalkyl
groups include
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H
/S\ A rO rS rNH 0
oxirane thiarane aziridine oxetane thiatane azetidine tetrahydrofuran
(oxiranyl) )thiaranyl) (aziridinyl) (oxetanyl) (thiatanyl) (azetidinyl)
(tetrahydrofuranyl)
v v UU UU
tetrahydrothiaphene pyrrolidine tetrahydropyran tetrahydrothiopyran
(tetrahydrothiaphenyl) (pyrrolidinyl) (tetrahydropyranyl)
(tetrahydrothiopyranyl)
N CO) O N U O CSC C CS)
Spiperidine 1,4-dioxane 1,4-oxathiane morpholine 1,4-dithiane
(piperidinyl) (i,4-dioxanyl) (i,4-oxathianyl) (morpholinyl) (1,4-dithianyl)
CNJ CNJ O S N
N S U U U
H
piperazine 1,4-azathiane oxepane thiepane azepane
(piperazinyl) (1,4-azathianyl) (oxepanyl) (thiepanyl) (azepanyl)
0 U 0 U
O S NH S
1,4-dioxepane 1,4-oxathiepane 1,4-oxaazepane 1,4-dithiepane
(1,4-dioxypanyl) (1,4-oxathiepanyl) (1,4-oxaazepanyl) (1,4-dithiepanyl)
H
C) C)
NH NH NH
1,4-thieazapane 1,4-diazepane
(1,4-thieazapanyl) (1,4-diazepanyl) tropane
(tropanyl)
[00208] Examples of partially unsaturated heterocyclyl groups include
H
U U 011 U
3,4-dihydro-2H-pyran 5,6-dihydro-2H-pyran 2H-pyran 1,2,5,6-tetrahydropyridine
(3,4-dihyd ro-2H-pyranyl) (5,6-dihydro-2H-pyranyl) (2H-pyranyl) (1,2,5,6-
tetrahydropyridinyl)
[00209] Other illustrative examples of heterocyclo groups, also referred to as
non-
aromatic heterocycles, include:
0 0 0 0 0 0 0
S N U
N D
0 Oe >Oo.
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N A10
(N) N
H H Ol ' ( H
CON
ax) - 15-0 0 C'') CS)
N N
H , H or the like.
[00210] The term heteroalicyclic also includes all ring forms of the
carbohydrates,
including but not limited to the monosaccharides, the disaccharides and the
oligosaccharides.
[00211] The term "halo" or, alternatively, "halogen" means fluoro, chloro,
bromo and
iodo.
[00212] The terms "haloalkyl," and "haloalkoxy" include alkyl and alkoxy
structures that
are substituted with one or more halogens. In embodiments, where more than one
halogen is
included in the group, the halogens are the same or they are different. The
terms
"fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy groups,
respectively, in
which the halo is fluorine.
[00213] The term "heteroalkyl" include optionally substituted alkyl, alkenyl
and alkynyl
radicals which have one or more skeletal chain atoms selected from an atom
other than
carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations
thereof. In
certain embodiments, the heteroatom(s) is placed at any interior position of
the heteroalkyl
group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -
CH2-NH-
CH3, -CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-
CH3, -CH2-S-CH2-CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -
Si(CH3)3, -CH2-CH=N-OCH3, and -CH=CH-N(CH3)-CH3. In some embodiments, up to
two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3 and -
CHz-
O-Si(CH3)3.
[00214] A "cyano" group refers to a CN group.
[00215] An "isocyanato" group refers to a NCO group.
[00216] A "thiocyanato" group refers to a CNS group.
[00217] An "isothiocyanato" group refers to a NCS group.
[00218] "Alkoyloxy" refers to a RC(=O)O- group.
[00219] "Alkoyl" refers to a RC(=O)- group.
Synthesis of Compounds
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[00220] In some embodiments, compounds of Formula I, II, III, IV, V, Va, or Vb
are
synthesized according to procedures described in Scheme 1 and in the Examples
section.
Scheme 1
B,
I NICIII
" " \ ~ \ \ \ \
Br Br
S N N O S' N N O N O
H H S N N O O O
II O IV
III
R3 (R4) R3
(R5)s ^~ ^
NH2 B r % M\
(R5s 1 6 / f~ i \ V III~ _ T
(RS% s-^ 1 (R )s
V \~H N N O H N N O
VI O O
IX
R3
QH
\ \ B`OH Br -O
(RS)s J (R )s
GN H N N O X
O
VII
[00221] Generally, compounds of Formula IX described herein are synthesized by
conversion of (methylthio)-pyridopyrimidinone, I, to its bromo derivative II.
Substitution at
the NH of the core, for example by alkylation with a halogen containing Q
forms substituted
compound III. Oxidation of the sulfanyl compound III using an oxidizing agent
such as for
example, chloroperbenzoic acid gives sulfinyl compound IV. Addition of the B-
ring (V)
results in compounds of Formula VI. Addition of the T ring (VIII) where M
represents a
group such a boronic acid, boronic ester,alkyl tin, zinc atom or other similar
moieties
generates compound IX. Alternatively, VI can be converted to its boronic acid
VIII and
ring T (X) can be attached via a halogen atom to generate IX. The procedures
described
herein are given merely as an example and should in no way limit the methods
of making
the compounds described herein.
Methods
[00222] Provided herein are methods for treating CNS disorders comprising
administration of a therapeutically effective amount of a p21-activated kinase
inhibitor (e.g.,
a compound of Formula I-XV) to an individual in need thereof. In some
embodiments of the
methods provided herein, administration of a p21-activated kinase inhibitor
alleviates or
reverses one or more behavioral symptoms (e.g., social withdrawal,
depersonalization, loss
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of appetite, loss of hygiene, delusions, hallucinations, depression, blunted
affect, avolition,
anhedonia, alogia, the sense of being controlled by outside forces or the
like) of the CNS
disorder (e.g. negative symptoms of schizophrenia). In some embodiments of the
methods
provided herein, administration of a p21-activated kinase inhibitor (e.g., a
compound of
Formula I-XV) alleviates or reverses one or more negative symptoms and/or
cognition
impairment associated with a CNS disorder (e.g., impairment in executive
function,
comprehension, inference, decision-making, planning, learning or memory
associated with
schizophrenia, Alzheimer's disease, FXS, autism or the like).
[00223] Also provided herein are methods for modulation of dendritic spine
morphology
and/or synaptic function comprising administering to an individual in need
thereof (e.g., an
individual suffering from or suspected of having schizophrenia, Parkinson's
disease,
Alzheimer's disease, epilepsy or the like) a therapeutically effective amount
of a PAK
inhibitor (e.g., a compound of Formula I-XV). In some embodiments, modulation
of
dendritic spine morphology and/or synaptic function alleviates or reverses
negative
symptoms and/or cognitive impairment associated with a CNS disorder. In some
embodiments, modulation of dendritic spine morphology and/or synaptic function
halts or
delays further deterioration of symptoms associated with a CNS disorder (e.g.,
progression
of cognitive impairments and/or loss of bodily functions). In some
embodiments,
modulation of dendritic spine morphology and/or synaptic function stabilizes
or reverses
symptoms of disease (e.g., reduces frequency of epileptic seizures, stabilizes
mild cognitive
impairment and prevents progression to early dementia). In some embodiments of
the
methods provided herein, administration of a p21-activated kinase inhibitor
halts or delays
progressive loss of memory and/or cognition associated with a CNS disorder
(e.g.,
Alzheimer's disease).
[00224] Provided herein are methods for modulation of synaptic function or
synaptic
plasticity comprising administering to an individual in need thereof (e.g., an
individual
suffering from or suspected of having any CNS disorder described herein) a
therapeutically
effective amount of a PAK inhibitor (e.g., a compound of Formula I-XV).
Modulation of
synaptic function or plasticity includes, for example, alleviation or reversal
of defects in
LTP, LTD or the like.
[00225] Defects in LTP include, for example, an increase in LTP or a decrease
in LTP in
any region of the brain in an individual suffering from or suspected of having
a CNS
disorder. Defects in LTD include for example a decrease in LTD or an increase
in LTD in
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any region of the brain (e.g., the temporal lobe, parietal lobe, the frontal
cortex, the
cingulate gyrus, the prefrontal cortex, the cortex, or the hippocampus or any
other region in
the brain or a combination thereof) in an individual suffering from or
suspected of having a
CNS disorder.
[00226] In some embodiments of the methods, administration of a PAK inhibitor
(e.g., a
compound of Formula I-XV) modulates synaptic function (e.g., synaptic
transmission
and/or plasticity) by increasing long term potentiation (LTP) in an individual
suffering from
or suspected of having a CNS disorder. In some embodiments of the methods
described
herein, administration of a PAK inhibitor (e.g., a compound of Formula I-XV)
to an
individual in need thereof modulates synaptic function (e.g., synaptic
transmission and/or
plasticity) by increasing long term potentiation (LTP) in the prefrontal
cortex, or the cortex,
or the hippocampus or any other region in the brain or a combination thereof
In some
embodiments of the methods described herein, administration of a PAK inhibitor
modulates
synaptic function (e.g., synaptic transmission and/or plasticity) by
decreasing long term
depression (LTD) in an individual suffering from or suspected of having a CNS
disorder. In
some embodiments of the methods described herein, administration of a PAK
inhibitor to an
individual in need thereof modulates synaptic function (e.g., synaptic
transmission and/or
plasticity) by decreasing long term depression (LTD) in the temporal lobe,
parietal lobe, the
frontal cortex, the cingulate gyrus, the prefrontal cortex, the cortex, or the
hippocampus or
any other region in the brain or a combination thereof.
[00227] In some embodiments of the methods described herein, administration of
a PAK
inhibitor reverses defects in synaptic function (i.e. synaptic transmission
and/or synaptic
plasticity, induced by soluble Abeta dimers or oligomers. In some embodiments
of the
methods described herein, administration of a PAK inhibitor reverses defects
in synaptic
function (i.e. synaptic transmission and/or synaptic plasticity, induced by
insoluble Abeta
oligomers and/or Abeta-containing plaques.
[00228] Provided herein are methods for stabilization of synaptic plasticity
comprising
administering to an individual in need thereof (e.g., an individual suffering
from or
suspected of having a CNS disorder) a therapeutically effective amount of a
PAK inhibitor
(e.g., a compound of Formula I-XV). In some embodiments of the methods
described
herein, administration of a PAK inhibitor stabilizes LTP or LTD following
induction (e.g.,
by theta-burst stimulation, high-frequency stimulation for LTP, low-frequency
(e.g., 1 Hz)
stimulation for LTD).
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[00229] Provided herein are methods for stabilization of synaptic transmission
comprising administering to an individual in need thereof (e.g., an individual
suffering from
or suspected of having a CNS disorder) a therapeutically effective amount of a
PAK
inhibitor (e.g., a compound of Formula I-XV). In some embodiments of the
methods
described herein, administration of a PAK inhibitor stabilizes LTP or LTD
following
induction (e.g., by theta-burst stimulation, high-frequency stimulation for
LTP, low-
frequency (e.g., 1 Hz) stimulation for LTD).
[00230] Also provided herein are methods for alleviation or reversal of
cortical
hypofrontality during performance of a cognitive task comprising administering
to an
individual in need thereof (e.g., an individual suffering from or suspected of
having a CNS
disorder) a therapeutically effective amount of a PAK inhibitor (e.g., a
compound of
Formula I-XV). In some embodiments of the methods described herein,
administration of a
PAK inhibitor to an individual suffering from or suspected of having a CNS
disorder
alleviates deficits in the frontal cortex, for example deficits in frontal
cortical activation,
during the performance of a cognitive task (e.g., a Wisconsin Card Sort test,
Mini-Mental
State Examination (MMSE), MATRICS cognitive battery, BACS score, Alzheimer's
disease Assessment Scale - Cognitive Subscale (ADAS-Cog), Alzheimer's disease
Assessment Scale - Behavioral Subscale (ADAS-Behav), Hopkins Verbal Learning
Test-
Revised or the like) and improves cognition scores of the individual.
[00231] Provided herein are methods for reversing abnormalities in dendritic
spine
morphology or synaptic function that are caused by mutations in high-risk
genes (e.g.
mutations in Amyloid Precursor Protein (APP), mutations in presenilin 1 and 2,
the epsilon4
allele, the 91bp allele in the telomeric region of 12q, Apolipoprotein E-4
(APOE4) gene,
SORL1 gene, reelin gene, DISCI gene, or any other high-risk allele) comprising
administering to an individual in need thereof a therapeutically effective
amount of a PAK
inhibitor (e.g., a compound of Formula I-XV). In some embodiments of the
methods
described herein, prophylactic administration of a PAK inhibitor to an
individual at a high
risk for developing a CNS disorder (e.g., a mutation in a DISCI gene pre-
disposes the
individual to schizophrenia, a mutation in an APOE4 gene pre-disposes the
individual to
Alzheimer's disease) reverses abnormalities in dendritic spine morphology
and/or synaptic
function and prevents development of the CNS disorder.
[00232] Provided herein are methods for stabilizing, reducing or reversing
abnormalities
in dendritic spine morphology or synaptic function that are caused by
increased activation
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of PAK at the synapse, comprising administration of a therapeutically
effective amount of a
PAK inhibitor (e.g., a compound of Formula I-XV) to an individual in need
thereof (e.g., an
individual suffering from or suspected of having a CNS disorder). In some
embodiments of
the methods described herein, increased activation of PAK at the synapse is
caused by
Abeta. In some instances, increased activation of PAK at the synapse is caused
by
redistribution of PAK from the cytosol to the synapse. In some embodiments of
the methods
described herein, administration of a therapeutically effective amount of a
PAK inhibitor
(e.g., a compound of Formula I-XV) to an individual in need thereof (e.g., an
individual
suffering from or suspected of having a CNS disorder) reduces or prevents
redistribution of
PAK from the cytosol to the synapse in neurons, thereby stabilizing, reducing
or reversing
abnormalities in dendritic spine morphology or synaptic function that are
caused by
increased activation of PAK at the synapse.
[00233] Provided herein are methods for delaying the onset of a CNS disorder
comprising administering to an individual in need thereof (e.g., an individual
with a high-
risk allele for a NC) a therapeutically effective amount of a PAK inhibitor
(e.g., a
compound of Formula I-XV). Provided herein are methods for delaying the loss
of dendritic
spine density comprising administering to an individual in need thereof (e.g.,
an individual
with a high-risk allele for a CNS disorder) a therapeutically effective amount
of a PAK
inhibitor. Provided herein are methods for modulation of spine density, shape,
spine length,
spine head volume, or spine neck diameter or the like comprising administering
to an
individual in need thereof (e.g., an individual suffering from or suspected of
having a CNS
disorder) a therapeutically effective amount of a PAK inhibitor (e.g., a
compound of
Formula I-XV). Provided herein are methods of modulating the ratio of mature
dendritic
spines to immature dendritic spines comprising administering to an individual
in need
thereof (e.g., an individual suffering from or suspected of having a CNS
disorder) a
therapeutically effective amount of a PAK inhibitor. Provided herein are
methods of
modulating the ratio of dendritic spines head volume to dendritic spines
length comprising
administering to an individual in need thereof (e.g., an individual suffering
from or
suspected of having a CNS disorder) a therapeutically effective amount of a
PAK inhibitor
(e.g., a compound of Formula I-XV).
[00234] In some embodiments of the methods described herein, administration of
a PAK
inhibitor (e.g., a maintenance dose of a PAK inhibitor) reduces the incidence
of recurrence
of one or more symptoms or pathologies in an individual (e.g., recurrence of
psychotic
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episodes, epileptic seizures or the like). In some embodiments of the methods
described
herein, administration of a PAK inhibitor causes substantially complete
inhibition of PAK
and restores dendritic spine morphology and/or synaptic function to normal
levels. In some
embodiments of the methods described herein, administration of a PAK inhibitor
causes
partial inhibition of PAK and restores dendritic spine morphology and/or
synaptic function
to normal levels.
[00235] Provided herein are methods for stabilizing, reducing or reversing
neuronal
withering and/or atrophy or nervous tissue and/or degeneration of nervous
tissue that is
associated with a CNS disorder. In some embodiments of the methods described
herein,
administration of a PAK inhibitor to an individual suffering from or suspected
of having a
CNS disorder (e.g., Alzheimer's disease, Parkinson's disease or the like)
stabilizes,
alleviates or reverses neuronal withering and /or atrophy and/or degeneration
in the
temporal lobe, parietal lobe, the frontal cortex, the cingulate gyrus or the
like. In some
embodiments of the methods described herein, administration of a PAK inhibitor
to an
individual suffering from or suspected of having a CNS disorder stabilizes,
reduces or
reverses deficits in memory and/or cognition and/or control of bodily
functions.
[00236] In some instances, a CNS disorder is associated with a decrease in
dendritic
spine density. In some embodiments of the methods described herein,
administration of a
PAK inhibitor increases dendritic spine density. In some instances, a CNS
disorder is
associated with an increase in dendritic spine length. In some embodiments of
the methods
described herein, administration of a PAK inhibitor decreases dendritic spine
length. In
some instances, a CNS disorder is associated with a decrease in dendritic
spine neck
diameter. In some embodiments of the methods described herein, administration
of a PAK
inhibitor increases dendritic spine neck diameter. In some instances, a CNS
disorder is
associated with a decrease in dendritic spine head diameter and/or dendritic
spine head
surface area and/or dendritic spine head volume. In some embodiments of the
methods
described herein, administration of a PAK inhibitor increases dendritic spine
head diameter
and/or dendritic spine head volume and/or dendritic spine head surface area.
[00237] In some instances, a CNS disorder is associated with an increase in
immature
spines and a decrease in mature spines. In some embodiments of the methods
described
herein, administration of a PAK inhibitor modulates the ratio of immature
spines to mature
spines. In some instances, a CNS disorder is associated with an increase in
stubby spines
and a decrease in mushroom-shaped spines. In some embodiments of the methods
described
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herein, administration of a PAK inhibitor modulates the ratio of stubby spines
to
mushroom-shaped spines.
[00238] In some embodiments of the methods described herein, administration of
a PAK
inhibitor modulates a spine:head ratio, e.g., ratio of the volume of the spine
to the volume of
the head, ratio of the length of a spine to the head diameter of the spine,
ratio of the surface
area of a spine to the surface area of the head of a spine, or the like,
compared to a
spine:head ratio in the absence of a PAK inhibitor. In certain embodiments, a
PAK inhibitor
suitable for the methods described herein modulates the volume of the spine
head, the width
of the spine head, the surface area of the spine head, the length of the spine
shaft, the
diameter of the spine shaft, or a combination thereof In some embodiments,
provided
herein is a method of modulating the volume of a spine head, the width of a
spine head, the
surface area of a spine head, the length of a spine shaft, the diameter of a
spine shaft, or a
combination thereof, by contacting a neuron comprising the dendritic spine
with an
effective amount of a PAK inhibitor described herein. In specific embodiments,
the neuron
is contacted with the PAK inhibitor in vivo.
[00239] Also described herein are methods for treating cancer in a subject
comprising
administering to a subject in need thereof a therapeutically effective amount
of a compound
of Formula I-XV. As used herein, "cancer" includes any malignant growth or
tumor caused
by abnormal and uncontrolled cell division. Examples of cancers include
pancreatic cancer,
gastrointestinal stromal tumors, lung cancer, stomach cancer, brain cancer,
kidney cancer,
breast cancer, head and neck cancer, myeloma, leukemia, lymphoma,
adenocarcinoma,
melanoma or the like.
[00240] In one embodiment is a method for treating cancer in a subject
comprising
administering to a subject in need thereof a therapeutically effective amount
of a compound
of Formula I wherein the cancer is selected from ovarian, breast, colon,
brain,
neurofibromatosis, CML, renal cell carcinoma, gastric, leukemia, NSCLC, CNS,
melanoma,
prostate, T-cell lymphoma, heptocellular, bladder and glioblastoma. In one
embodiment,
the breast cancer is tamoxifen-resistant or intolerant breast cancer. In
another embodiment,
the CML is imatinib resistant or intolerant CML.
[00241] In one embodiment, is a method for modulating a p21 activated kinase
comprising contacting a compound of Formula I-XV with a p21 activated kinase
such that
PAK expression or activation has been altered. PAK kinases have been
identified as key
regulators of cancer-cell signaling networks where they regulate essential
biological
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processes. These processes include cytoskeletal dynamics, energy homeostasis,
cell
survival, differentiation, anchorage-independent growth, mitosis, and hormone
dependence.
Dysregulation of these processes by alterations in PAK expression or
activation have been
reported in numerous human cancers. See, e.g., Kumar R, Gururaj AE, Barnes CJ,
p21-
activated kinases in cancer, Nat Rev Cancer, 2006; 6: 459-471, which is
incorporated by
reference herein to the extent it is relevant.
[00242] In another embodiment is a method for treating cancer in a subject
comprising
administering to a subject in need thereof a therapeutically effective amount
of a compound
of Formula I-XV wherein the cancer is selected from pancreatic cancer,
gastrointestinal
stromal tumors, lung cancer, stomach cancer, brain cancer, kidney cancer,
breast cancer,
head and neck cancer, myeloma, leukemia, lymphoma, adenocarcinoma, bone
cancer,
cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal
cancer, cancer of
the anal region, stomach cancer, colon cancer, carcinoma of the fallopian
tubes, carcinoma
of the endometrium, carcinoma of the cervix, carcinoma of the vagina,
carcinoma of the
vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small
intestine, cancer of
the endocrine system, cancer of the thyroid gland, cancer of the parathyroid
gland, cancer of
the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of
the penis, prostate
cancer, lymphocytic lymphomas, cancer of the bladder, renal cell carcinoma,
carcinoma of
the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS
lymphoma,
spinal axis tumors, brain stem glioma, pituitary adenoma, or a combination of
one or more
of the foregoing cancers.
[00243] In certain embodiments, a compound or a composition comprising a
compound
described herein is administered for prophylactic and/or therapeutic
treatments. In
therapeutic applications, the compositions are administered to an individual
already
suffering from a disease or condition, in an amount sufficient to cure or at
least partially
arrest the symptoms of the disease or condition. In various instances, amounts
effective for
this use depend on the severity and course of the disease or condition,
previous therapy, an
individual's health status, weight, and response to the drugs, and the
judgment of the
treating physician.
[00244] In some embodiments, a composition containing a therapeutically
effective
amount of a PAK inhibitor is administered prophylactically to an individual
that while not
overtly manifesting symptoms of a CNS disorder has been identified as having a
high risk
of developing a CNS disorder, e.g., an individual is identified as being a
carrier of a
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mutation or polymorphism associated with a higher risk to develop a CNS
disorder (see,
e.g., Hall et al (2006), Nat Neurosci., 9(12):1477-8), or an individual that
is from a family
that has a high incidence of CNS disorders. In some embodiments, MRI is used
to detect
brain morphological changes in individuals prior to the onset of disease (see,
e.g., Toga et al
(2006), TINS, 29(3):148-159). For example, in some instances, the typical age
of onset for
schizophrenia is post-puberty. In some instances, the typical age of onset for
schizophrenia
is between 20-28 for males and 26-32 for females. For example, in some
instances, a typical
age of onset for Alzheimer's disease is about 55 -80 years. Accordingly, in
some
embodiments, a PAK inhibitor is administered prophylactically to an individual
at risk
between about Ito about 10 years, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years
prior to an
established and/or typical age range of onset for a CNS disorder.
[00245] In prophylactic applications, compounds or compositions containing
compounds
described herein are administered to an individual susceptible to or otherwise
at risk of a
particular disease, disorder or condition. In certain embodiments of this use,
the precise
amounts of compound administered depend on an individual's state of health,
weight, and
the like. Furthermore, in some instances, when a compound or composition
described herein
is administered to an individual, effective amounts for this use depend on the
severity and
course of the disease, disorder or condition, previous therapy, an
individual's health status
and response to the drugs, and the judgment of the treating physician.
[00246] In certain instances, wherein following administration of a selected
dose of a
compound or composition described herein, an individual's condition does not
improve,
upon the doctor's discretion the administration of a compound or composition
described
herein is optionally administered chronically, that is, for an extended period
of time,
including throughout the duration of an individual's life in order to
ameliorate or otherwise
control or limit the symptoms of an individual's disorder, disease or
condition.
[00247] In certain embodiments, an effective amount of a given agent varies
depending
upon one or more of a number of factors such as the particular compound,
disease or
condition and its severity, the identity (e.g., weight) of an individual or
host in need of
treatment, and is determined according to the particular circumstances
surrounding the case,
including, e.g., the specific agent being administered, the route of
administration, the
condition being treated, and an individual or host being treated. In some
embodiments,
doses administered include those up to the maximum tolerable dose. In certain
embodiments, about 0.02 to about 5000 mg per day, from about Ito about 1500 mg
per
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day, about 1 to about 100 mg/day, about 1 to about 50 mg/day, or about 1 to
about 30
mg/day, or about 5 to about 25 mg/day of a compound described herein is
administered. In
various embodiments, the desired dose is conveniently be presented in a single
dose or in
divided doses administered simultaneously (or over a short period of time) or
at appropriate
intervals, for example as two, three, four or more sub-doses per day.
[00248] In certain instances, there are a large number of variables in regard
to an
individual treatment regime, and considerable excursions from these
recommended values
are considered within the scope described herein. Dosages described herein are
optionally
altered depending on a number of variables such as, by way of non-limiting
example, the
activity of the compound used, the disease or condition to be treated, the
mode of
administration, the requirements of an individual, the severity of the disease
or condition
being treated, and the judgment of the practitioner.
[00249] Toxicity and therapeutic efficacy of such therapeutic regimens are
optionally
determined by pharmaceutical procedures in cell cultures or experimental
animals,
including, but not limited to, the determination of the LD50 (the dose lethal
to 50% of the
population) and the ED50 (the dose therapeutically effective in 50% of the
population). The
dose ratio between the toxic and therapeutic effects is the therapeutic index
and it can be
expressed as the ratio between LD50 and ED50. Compounds exhibiting high
therapeutic
indices are preferred. In certain embodiments, data obtained from cell culture
assays and
animal studies are used in formulating a range of dosage for use in human. In
specific
embodiments, the dosage of compounds described herein lies within a range of
circulating
concentrations that include the ED50 with minimal toxicity. The dosage
optionally varies
within this range depending upon the dosage form employed and the route of
administration
utilized.
Combination Therapy
[00250] In some embodiments, one or more PAK inhibitors are used in
combination with
one or more other therapeutic agents to treat an individual suffering from a
CNS disorder.
The combination of PAK inhibitors with a second therapeutic agent (e.g., a
typical or
atypical antipsychotic agent, an mG1uR1 antagonist, an mG1uR5 antagonist, an
mG1uR5
potentiator, a mGluR2 agonist, an alpha7 nicotinic receptor agonist or
potentiator, an
antioxidant, a neuroprotectant, a trophic factor, an anticholinergic, a beta-
secretase inhibitor
or the like) allows a reduced dose of both agents to be used thereby reducing
the likelihood
of side effects associated with higher dose monotherapies. In one embodiment,
the dose of a
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second active agent is reduced in the combination therapy by at least 50%
relative to the
corresponding monotherapy dose, whereas the PAK inhibitor dose is not reduced
relative to
the monotherapy dose; in further embodiments, the reduction in dose of a
second active
agent is at least 75%; in yet a further embodiment, the reduction in dose of a
second active
agent is at least 90%. In some embodiments, the second therapeutic agent is
administered at
the same dose as a monotherapy dose, and the addition of a PAK inhibitor to
the treatment
regimen alleviates symptoms of a CNS disorder that are not treated by
monotherapy with
the second therapeutic agent. Symptoms and diagnostic criteria for all of the
conditions
mentioned above are described in detail in the Diagnostic and Statistical
Manual of Mental
Disorders, fourth edition, American Psychiatric Association (2005) (DSM-IV).
[00251] In some embodiments, the combination of a PAK inhibitor and a second
therapeutic agent is synergistic (e.g., the effect of the combination is
better than the effect of
each agent alone). In some embodiments, the combination of a PAK inhibitor and
a second
therapeutic agent is additive (e.g., the effect of the combination of active
agents is about the
same as the effect of each agent alone). In some embodiments, an additive
effect is due to
the PAK inhibitor and the second therapeutic agent modulating the same
regulatory
pathway. In some embodiments, an additive effect is due to the PAK inhibitor
and the
second therapeutic agent modulating different regulatory pathways. In some
embodiments,
an additive effect is due to the PAK inhibitor and the second therapeutic
agent treating
different symptom groups of the CNS disorder (e.g., a PAK inhibitor treats
negative
symptoms and the second therapeutic agent treats positive symptoms of
schizophrenia). In
some embodiments, administration of a second therapeutic agent treats the
remainder of the
same or different symptoms or groups of symptoms that are not treated by
administration of
a PAK inhibitor alone.
[00252] In some embodiments, administration of a combination of a PAK
inhibitor and a
second therapeutic agent alleviates side effects that are caused by the second
therapeutic
agent (e.g., side effects caused by an antipsychotic agent or a nootropic
agent). In some
embodiments, administration of the second therapeutic agent inhibits
metabolism of an
administered PAK inhibitor (e.g., the second therapeutic agent blocks a liver
enzyme that
degrades the PAK inhibitor) thereby increasing efficacy of a PAK inhibitor. In
some
embodiments, administration of a combination of a PAK inhibitor and a second
therapeutic
agent (e.g. a second agent that modulates dendritic spine morphology (e.g.,
minocyline))
improves the therapeutic index of a PAK inhibitor.
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Agents for Treating Psychotic Disorders
[00253] Where a subject is suffering from or at risk of suffering from a
psychotic
disorder (e.g., schizophrenia), a PAK inhibitor composition described herein
is optionally
used together with one or more agents or methods for treating a psychotic
disorder in any
combination. Alternatively, a PAK inhibitor composition described herein is
administered
to a patient who has been prescribed an agent for treating a psychotic
disorder. In some
embodiments, administration of a PAK inhibitor in combination with an
antipsychotic agent
has a synergistic effect and provides an improved therapeutic outcome compared
to
monotherapy with antipsychotic agent or monotherapy with PAK inhibitor.
Alternatively, a
PAK inhibitor composition described herein is administered to a patient who is
non-
responsive to, or being unsatisfactorily treated with an antipsychotic agent.
[00254] In some embodiments, a PAK inhibitor composition described herein is
administered in combination with an antipsychotic having 5-HT2A antagonist
activity. In
some embodiments, a PAK inhibitor composition described herein is administered
in
combination with a selective 5-HT2A antagonist.
[00255] Examples of therapeutic agents/treatments for treating a psychotic
disorder
include, but are not limited to, any of the following: typical antipsychotics,
e.g.,
Chlorpromazine (Largactil, Thorazine), Fluphenazine (Prolixin), Haloperidol
(Haldol,
Serenace), Molindone, Thiothixene (Navane), Thioridazine (Mellaril),
Trifluoperazine
(Stelazine), Loxapine, Perphenazine, Prochlorperazine (Compazine, Buccastem,
Stemetil),
Pimozide (Orap), Zuclopenthixol; and atypical antipsychotics, e.g., LY2140023,
Clozapine,
Risperidone, Olanzapine, Quetiapine, Ziprasidone, Aripiprazole, Paliperidone,
Asenapine,
Iloperidone, Sertindole, Zotepine, Amisulpride, Bifeprunox, and Melperone.
Agents for Treating Mood Disorders
[00256] Where a subject is suffering from or at risk of suffering from a mood
disorder
(e.g., clinical depression), a PAK inhibitor composition described herein is
optionally used
together with one or more agents or methods for treating a mood disorder in
any
combination. Alternatively, a PAK inhibitor composition described herein is
administered
to a patient who has been prescribed an agent for treating a mood disorder.
Alternatively, a
PAK inhibitor composition described herein is administered to a patient who is
non-
responsive to or being unsatisfactorily treated with an agent for treating a
mood disorder.
[00257] Examples of therapeutic agents/treatments for treating a mood disorder
include,
but are not limited to, any of the following: selective serotonin reuptake
inhibitors (SSRIs)
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such as citalopram (Celexa), escitalopram (Lexapro, Esipram), fluoxetine
(Prozac),
paroxetine (Paxil, Seroxat), sertraline (Zoloft), fluvoxamine (Luvox);
serotonin-
norepinephrine reuptake inhibitors (SNRIs) such as venlafaxine (Effexor),
desvenlafaxine,
nefazodone, milnacipran, duloxetine (Cymbalta), bicifadine; tricyclic
antidepressants such
as amitriptyline, amoxapine, butriptyline, clomipramine, desipramine,
dosulepin, doxepin,
impramine, lofepramine, nortriptyline; monoamine oxidase inhibitors (MAOIs)
such as
isocarboxazid, linezolid, moclobemide, nialamide, phenelzine, selegiline,
tranylcypromine,
trimipramine; and other agents such as mirtazapine, reboxetine, viloxazine,
malprotiline,
and bupropion.
Agents for Treating Epilepsy
[00258] Where a subject is suffering from or at risk of suffering from
epilepsy, a PAK
inhibitor composition described herein is optionally used together with one or
more agents
or methods for treating epilepsy in any combination. Alternatively, a PAK
inhibitor
composition described herein is administered to a patient who has been
prescribed an agent
for treating epilepsy. Alternatively, a PAK inhibitor composition described
herein is
administered to a patient who is refractory to or being unsatisfactorily
treated with an agent
for treating epilepsy.
[00259] Examples of therapeutic agents/treatments for treating epilepsy
include, but are
not limited to, any of the following: carbamazepine, clobazam, clonazepam,
ethosuximide,
felbamate, fosphenytoin, gabapentin, lamotrigine, levetiracetam,
oxcarbazepine,
phenobarbital, phenytoin, pregabalin, primidone, sodium valproate, tiagabine,
topiramate,
valproate semisodium, valproic acid, vigabatrin, and zonisamide.
Agents for Treating Huntington's Disease
[00260] Where a subject is suffering from or at risk of suffering from
Huntington's
disease, a PAK inhibitor composition described herein is optionally used
together with one
or more agents or methods for treating Huntington's disease in any
combination.
Alternatively, a PAK inhibitor composition described herein is administered to
a patient
who has been prescribed an agent for treating Huntington's disease.
Alternatively, a PAK
inhibitor composition described herein is administered to a patient who is
refractory to or
being unsatisfactorily treated with an agent for treating Huntington's
disease.
[00261] Examples of therapeutic agents/treatments for treating Huntington's
disease
include, but are not limited to, any of the following: omega-3 fatty acids,
miraxion,
Haloperidol, dopamine receptor blockers, creatine, cystamine, cysteamine,
clonazepam,
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clozapine, Coenzyme Q10, minocycline, antioxidants, antidepressants (notably,
but not
exclusively, selective serotonin reuptake inhibitors SSRIs, such as
sertraline, fluoxetine, and
paroxetine), select dopamine antagonists, such as tetrabenazine; and RNAi
knockdown of
mutant huntingtin (mHtt).
Agents for Treating Parkinson's Disease
[00262] Where a subject is suffering from or at risk of suffering from
Parkinson's
Disease, a PAK inhibitor composition described herein is optionally used
together with one
or more agents or methods for treating Parkinson's disease in any combination.
Alternatively, a PAK inhibitor composition described herein is administered to
a patient
who has been prescribed an agent for treating Parkinson's disease.
Alternatively, a PAK
inhibitor composition described herein is administered to a patient who is
refractory to or
being unsatisfactorily treated with an agent for treating Parkinson's disease.
[00263] Examples of therapeutic agents/treatments for treating Parkinson's
Disease
include, but are not limited to any of the following: L-dopa, carbidopa,
benserazide,
tolcapone, entacapone, bromocriptine, pergolide, pramipexole, ropinirole,
cabergoline,
apomorphine, lisuride, selegiline, or rasagiline.
Group I mG1uR antagonists
[00264] In some embodiments, one or more PAK inhibitors are used in
combination with
one or more Group I metabotropic glutamate receptor (mGluR) antagonists (e.g.,
mGluR5
antagonists) to treat an individual suffering from a CNS disorder. The
combination of PAK
inhibitors with Group I mGluR antagonists allows a reduced dose of both agents
to be used
thereby reducing the likelihood of side effects associated with higher dose
monotherapies.
[00265] In some embodiments, reduction of signaling from a Group I mGluR
(mGluR5)
in vivo by genetic engineering (using mGluR5 knock-out heterozygote animals)
leads to a
reversal of the dendritic spine and behavioral defects. In some instances,
where an
individual is suffering from or at risk of suffering from a CNS disorder, a
PAK inhibitor
composition described herein is optionally used together with one or Group I
mG1uR
antagonists. Group I mGluR antagonists include antagonists that are mGluRI -
selective
antagonists, mG1uR5-selective antagonists, or antagonists that antagonize both
mG1uR1 and
mGluR5. In some embodiments, a PAK inhibitor composition is used in
combination with
an mG1uR5-selective antagonist. In some embodiments, a PAK inhibitor
composition is
used in combination with an mG1uR1-selective antagonist. In some embodiments,
a PAK
inhibitor composition is used in combination with a Group I mGluR antagonist
that
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antagonizes both mG1uR1 and mG1uR5 (i.e., an antagonist that is not selective
for mG1uR1
or mG1uR5). As used herein, the term "selective antagonist" indicates that the
antagonist
has an ED50 for antagonizing a first receptor (e.g., mG1uR5) that is at least
about 10 fold to
about 1000 fold lower, e.g., 11, 20, 30, 40, 50, 100, 105, 125, 135, 150, 200,
300, 400, 500,
600, 700, 800, 900, or any other fold lower from about 10 fold to about 1000
fold lower
than the ED50 for antagonism of a second receptor (e.g., mG1uR1).
[00266] Examples of Group I mGluR antagonists include, but are not limited to,
any of
the following (E)-6-methyl-2-styryl-pyridine (SIB 1893), 6-methyl-2-
(phenylazo)-3-
pyridinol,.alpha.-methyl-4-carboxyphenylglycine (MCPG), or 2-methyl-6-
(phenylethynyl)-
pyridine (MPEP). Examples of Group I mGluR antagonists also include those
described in,
e.g., U.S. Patent Application Serial Nos: 10/076,618; 10/211,523; and
10/766,948.
Examples of mG1uR5-selective antagonists include, but are not limited to those
described
in, e.g., U.S. Patent No: 7,205,411 and U.S. Patent Application Serial No
11/523,873.
Examples of mG1uR1-selective antagonists include, but are not limited to,
those described
in, e.g., U.S. Patent No. 6,482,824.
[00267] In some embodiments, the mGluR Group I antagonist is AIDA (1-
aminoindan-
1,5-dicarboxylic acid); ACDPP (3-Amino-6-chloro-5-dimethylamino-N-2-
pyridinylpyrazinecarboxamide hydrochloride; DL-AP3 (DL-2-Amino-3-
phosphonopropionic acid); BAY-36-7620 ((3aS,6aS)-Hexahydro-5-methylene-6a-(2-
naphthalenylmethyl)-1H-cyclopenta[c]furan-l-one); Fenobam; 4 CPG ((S)4-
carboxyphenylglycine); (S)-4C3HPG ((S)-4-carboxy-3-hydroxyphenylglycine);
CPCCOEt
(7-hydroxyiminocyclopropan[b]chromen-la-carboxylic acid ethyl ester); LY
367385 ((S)-
(+)-a-Amino-4-carboxy-2-methylbenzeneacetic acid); LY 456236 hydrochloride (6-
methoxy-N-(4-methoxyphenyl) quinazolin-4-amine, MPMQ hydrochloride); 3-MATIDA
(a-Amino-5-carboxy-3-methyl-2-thiopheneacetic acid); MCPG (a-methyl-4-
carboxyphenylglycine); MPEP (2-methyl-6-(phenylethynyl)-pyridine); (MTEP) 3-
[(2-
methyl-1,3-thiazol-4-yl)ethynyl]-pyridine; PHCCC (N-Phenyl-7-
(hydroxyimino)cyclopropa[b]chromen-la-carbox amide; SIB 1757 (6-Methyl-2-
(phenylazo)-3-pyridinol; SIB 1893 (2-Methyl-6-(2-phenylethenyl)pyridine; YM
298198
hydrochloride (6-Amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-a]benzimidazole-2-
carboxamidehydrochloride); (YM-193167 (6-amino-N-cyclohexyl-N,3-
dimethylthiazolo[3,2-a]benzimidazole-2-carboxamide); (NPS 2390 (Quinoxaline-2-
carboxylic acid adamantan-1-ylamide); 3-(5-(pyridin-2-yl)-2H-tetrazol-2-
yl)benzonitrile; 3-
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[3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)phenyl]-4-methylpyridine; 3-
fluoro-5-(5-
pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile; N-cyclohexyl-6-{[(2-
methoxyethyl)(methyl)amino]methyl} -N-methylthiazolo [3,2-a]benzimidazole-2-
carboxamide (YM-202074); Desmethyl-YM298198 (6-Amino-N-cyclohexyl-3-
methylthiazolo[3,2-a]benzimidazole-2-carboxamide hydrochloride); MPEP
hydrochloride
(2-Methyl-6-(phenylethynyl)pyridine hydrochloride); (S)-MCPG ((S)-a-Methyl-4-
carboxyphenylglycine); (RS)-MCPG ((RS)-a-Methyl-4-carboxyphenylglycine); E4CPG
((RS)-a-Ethyl-4-carboxyphenylglycine); Hexylhomoibotenic acid (a-Amino-4-hexyl-
2,3-
dihydro-3-oxo-5-isoxazolepropanoic acid; HexylHIBO); (S)-Hexylhomoibotenic
acid ((S)-
a-Amino-4-hexyl-2,3-dihydro-3-oxo-5-isoxazolepropanoic acid; (S)-HexylHIBO);
EMQMCM (3-ethyl-2-methyl-quinolin-6-yl)-(4-methoxy-cyclohexyl)-methanone
methanesulfonate); JNJ 16259685; 8214127 (1-(3,4-dihydro-2H-pyrano[2,3-
b]quinolin-7-
yl)-2-phenyl-l-ethanone); (S)-3-Carboxy-4-hydroxyphenylglycine ((S)-3C4HPG);
Anti-
mGluS blocking peptide ([K]-SSPKYDTLIIRDYTQSSSSL); DFB (3,3'-
Difluorobenzaldazine); DMeOB ([(3-Methoxyphenyl)methylene]hydrazone-3-
methoxybenzaldehyde); Anti-mGlu5 (([K]-SSPKYDTLIIRDYTQSSSSL); reluzole ; or
combinations thereof.
[00268] In some embodiments, the modulator of a Group I mGluR is S-(4-Fluoro-
phenyl)- {3 -[3 -(4-fluoro-phenyl)-[ 1,2,4] oxadiazol-5-yl]-piperidin-1-yl} -
methanone
(ADX47273) (Positive allosteric modulator); 4-[1-(2-fluoropyridin-3-yl)-5-
methyl-lH-
1,2,3-triazol-4-yl]-N-isopropyl-N-methyl-3,6-dihydropyridine-1(2H)-carboxamide
(FTIDC); 6-(3-methoxy-4-(pyridin-2-yl)phenyl)imidazole[2,1-b]thiazole; 2-(2-
methoxy-4-
(4-(pyridin-2-yl)oxazol-2-yl)phenyl)acetonitrile; 2-(4-(benzo[d]oxazol-2-yl)-2-
methoxyphenyl)acetonitrile; 2-(4-(2,3-dihydro-lH-inden-2-ylamino)4a,5,6,7,8,8a-
hexahydroquinazolin-2ylthio)ethanol; or combinations thereof.
[00269] In some embodiments, where a Group I mGluR antagonist (e.g., an mG1uR5
antagonist) is administered in combination with a PAK inhibitor, the dose of
the Group I
mGluR antagonist ranges from about 0.001 mg/kg/day to about 30.0 mg/kg/day,
e.g., about
0.005 mg/kg/day, 0.009 mg/kg/day, 0.010 mg/kg/day, 0.050 mg/kg/day, 0.20
mg/kg/day,
0.50 mg/kg/day, 0.75 mg/kg/day, 1.0 mg/kg/day, 2.0 mg/kg/day, 3.5 mg/kg/day,
4.5
mg/kg/day,5.0 mg/kg/day, 6.2 mg/kg/day, 6.8 mg/kg/day, 7.0 mg/kg/day, 10.0
mg/kg/day,
15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, or any other dose from about 0.001
mg/kg/day
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to about 10.0 mg/kg/day, from about 0.001 mg/kg/day to about 20.0 mg/kg/day,
or from
about 0.01 mg/kg/day to about 20.0 mg/kg/day.
[00270] In some embodiments, the combination treatment comprises administering
a
combined dosage form that is a pharmacological composition comprising a
therapeutically
effective amount of a PAK inhibitor and a Group I mGluR antagonist (e.g., an
mG1uR5-
selective antagonist) as described herein. In some embodiments, the
pharmacological
composition comprises a PAK inhibitor compound and an mG1uR5-selective
antagonist
selected from U.S. Patent No: 7,205,411.
mGluR agonists
[00271] In some embodiments, a second therapeutic agent used in combination
with a
PAK inhibitor is a Group I mG1uR1 agonist. Examples of mG1uR1 agonists and/or
mG1uR1
potentiators include and are not limited to ACPT-I ((1S,3R,4S)-1-
aminocyclopentane-1,3,4-
tricarboxylic acid); L-AP4 (L-(+)-2-Amino-4-phosphonobutyric acid); (S)-3,4-
DCPG ((S)-
3,4-dicarboxyphenylglycine); (RS)-3,4-DCPG ((RS)-3,4-dicarboxyphenylglycine);
(RS)-4-
phosphonophenylglycine ((RS)PPG); AMN082 (,N'-bis(diphenylmethyl)-1,2-
ethanediamine
dihydrochloride); DCG-IV ((25,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine)
or the like.
In some embodiments, an mG1uR1 agonist is AMN082. In some embodiments, a
second
therapeutic agent is a mGluR2/3 agonist or mGluR2/3 potentiator. Examples of
mGluR2/3
agonists include and are not limited to LY389795 ((-)-2-thia-4-aminobicyclo-
hexane-4,6-
dicarboxylate); LY379268 ((-)-2-oxa-4-aminobicyclo-hexane-4,6-dicarboxylate);
LY354740 ((+)-2-aminobicyclo-hexane-2,6dicarboxylate); DCG-IV ((25,2'R,3'R)-2-
(2',3'-
dicarboxycyclopropyl)glycine); 2R,4R-APDC (2R,4R-4-aminopyrrolidine-2,4-
dicarboxylate), (S)-3C4HPG ((S)-3-carboxy-4-hydroxyphenylglycine); (S)-4C3HPG
((S)-4-
carboxy-3-hydroxyphenylglycine); L-CCG-I ((2S,1'S,2'S)-2-
(carboxycyclopropyl)glycine);
and/or combinations thereof. Examples of mGluR2 agonists or mGluR2
potentiators include
and are not limited to positive allosteric modulators of mGluR2, including
ADX71149
(Addex Partner). Examples of mGluR5 agonists or mGluR5 potentiators include
and are not
limited to MPEP, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), 1S,3R-1-amino-
l,3-
cyclopentanedicarboxylate (ACPD) or the like.
Apha7 nicotinic receptor modulators
[00272] In some embodiments, one or more PAK inhibitors are used in
combination with
one or more alpha7 nicotinic receptor modulators to treat an individual
suffering from a
CNS disorder. Alpha7 nicotinic receptor modulators include alpha7 nicotinic
receptor
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agonists, alpha7 nicotinic receptor antagonists, and/or alpha7 nicotinic
receptor modulators
positive allosteric potentiators. The combination of PAK inhibitors with
alpha7 nicotinic
receptor modulators allows a reduced dose of both agents to be used thereby
reducing the
likelihood of side effects associated with higher dose monotherapies.
[00273] Examples of alpha7 nicotinic receptor agonists include and are not
limited to
(+)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzo[b]furan- 2-carboxamide, PHA-709829,
PNU-
282,987, A-582941, TC-1698, TC-5619, GTS-21, SSR180711, tropisetron or the
like.
Examples of alpha7 nicotinic receptor antagonists include a.-conotoxin,
quinolizidine or the
like. Alpha7 nicotinic receptor allosteric potentiators include PNU-120596, NS-
1738,
l0 XY4083, A-867744, EVP-6124 (Envivo), or the like.
Cholinesterase inhibitors
[00274] Where a subject is suffering from or at risk of suffering from
Alzheimer's
disease, a PAK inhibitor composition described herein is optionally used
together with one
or more agents or methods for treating Alzheimer's disease in any combination.
In some
embodiments, a PAK inhibitor composition described herein is administered to a
patient
who has been prescribed an acetylcholinesterase inhibitor. In some
embodiments,
administration of a PAK inhibitor in combination with an acetylcholinesterase
inhibitor has
a synergistic effect and provides an improved therapeutic outcome compared to
monotherapy with acetylcholinesterase inhibitors or monotherapy with PAK
inhibitor.
Alternatively, a PAK inhibitor composition described herein is administered to
an
individual who is non-responsive to, or being unsatisfactorily treated with an
acetylcholinesterase inhibitor. Example of acetylcholinesterase inhibitors
include donepezil
(Aricept), galantamine (Razadyne), rivastigmine (Exelon and Exelon Patch).
Muscarinic modulators
[00275] In some embodiments, a PAK inhibitor composition described herein is
administered to a patient in combination with a muscarinic receptor modulator.
In some
embodiments, the muscarinic receptor modulator is a M1 muscarinic receptor
agonist. In
some embodiments, the muscarinic receptor modulator is AF 102B, AF 150(S),
AF267B, N-
{ 1-[3-(3-oxo-2,3-dihydrobenzo[1,4]oxazin-4-yl)propyl]piperidin-4-yl}-2-
phenylacetamide,
BRL-55473, NXS-292, NXS-267, MCD-386, AZD-6088, N-Desmethylclozapine or a
similar compound. In some embodiments, the muscarinic receptor modulator is a
positive
allosteric modulator of M1 muscarinic receptors. Examples of positive
allosteric M1
muscarinic receptor modulators include, but are not limited to, VUO119498,
VU0027414,
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VU0090157, VU0029767, BQCA, TBPB or 77-LH-28-1. In some embodiments, the
muscarinic receptor modulator is a M4 muscarinic receptor agonist. In some
embodiments,
the muscarinic receptor modulator is a positive allosteric modulator of M4
muscarinic
receptors. Examples for positive allosteric M4 muscarinic receptor modulators
include, but
are not limited to, VU0010010, VU0152099, VU0152100, or LY2033298.
NMDA receptor antagonists
[00276] Where a subject is suffering from or at risk of suffering from
Alzheimer's
disease, a PAK inhibitor composition described herein is optionally used
together with one
or more agents or methods for treating Alzheimer's disease in any combination.
In some
embodiments, a PAK inhibitor composition described herein is administered to a
patient
who has been prescribed an NMDA receptor antagonist. Examples of NMDA receptor
antagonists useful in the methods and compositions described herein include
and are not
limited to memantine.
Neuroprotectants
[00277] In some embodiments, a PAK inhibitor or a composition thereof
described
herein is administered in combination with a neuroprotectant such as, for
example,
minocycline, resveratrol or the like.
Trophic factors
[00278] In some embodiments, a PAK inhibitor or a composition thereof
described
herein is administered in combination with atrophic agent including, by way of
example,
glial derived nerve factor (GDNF), brain derived nerve factor (BDNF) or the
like.
Antioxidants
[00279] Where a subject is suffering from or at risk of suffering from a CNS
disorder
(e.g., Alzheimer's disease, Mild Cognitive Impairment), a PAK inhibitor
composition
described herein is optionally used together with one or more agents or
methods for treating
the CNS disorder in any combination. In some embodiments, a PAK inhibitor
composition
described herein is administered to a patient who is taking or has been
prescribed an
antioxidant. Examples of antioxidants useful in the methods and compositions
described
herein include and are not limited to ubiquinone, aged garlic extract,
curcumin, lipoic acid,
beta-carotene, melatonin, resveratrol, Ginkgo biloba extract, vitamin C,
viatmin E or the
like.
Metal Protein attenuating compounds
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[00280] Where a subject is suffering from or at risk of suffering from a CNS
disorder
(e.g., Alzheimer's disease, Parkinson's disease), a PAK inhibitor composition
described
herein is optionally used together with one or more agents or methods for
treating the CNS
disorder in any combination. In some embodiments, a PAK inhibitor composition
described
herein is administered to a patient who has been prescribed a Metal Protein
Attenuating
agent. Examples of Metal Protein Attenuating agents useful in the methods and
compositions described herein include and are not limited to 8-
Hydroxyquinoline,
iodochlorhydroxyquin or the like and derivatives thereof.
Beta-secretase inhibitors
[00281] Where a subject is suffering from or at risk of suffering from a CNS
disorder
(e.g., Alzheimer's disease), a PAK inhibitor composition described herein is
optionally used
together with one or more agents or methods for treating the CNS disorder in
any
combination. In some embodiments, a PAK inhibitor composition described herein
is
administered to a patient who has been prescribed a beta secretase inhibitor.
Examples of
beta secretase inhibitors useful in the methods and compositions described
herein include
and are not limited to LY450139, 2-Aminoquinazolines compounds described in J.
Med.
Chem. 50 (18): 4261-4264, beta secretase inhibitors described therein are
incorporated
herein by reference, or the like.
Gamma secretase inhibitors
[00282] Where a subject is suffering from or at risk of suffering from a CNS
disorder
(e.g., Alzheimer's disease), a PAK inhibitor composition described herein is
optionally used
together with one or more agents or methods for treating the CNS disorder in
any
combination. In some embodiments, a PAK inhibitor composition described herein
is
administered to a patient who has been prescribed a beta secretase inhibitor.
Examples of
beta secretase inhibitors useful in the methods and compositions described
herein include
and are not limited to LY-411575, (2S)-2-hydroxy-3-methyl-N-((1S)-1-methyl-2-
{[(1S)-3-
methyl-2-oxo-2,3,4,5-tetrahydro-1H--3 -benzazepin-1-yl]amino } -2-
oxoethyl)butanamide
(semagacestat), (R)-2-(3-Fluoro-4-phenylphenyl)propanoic acid (Tarenflurbil),
or the like.
Antibodies
[00283] Where a subject is suffering from or at risk of suffering from a CNS
disorder
(e.g., Alzheimer's disease), a PAK inhibitor composition described herein is
optionally used
together with one or more agents or methods for treating the CNS disorder in
any
combination. In some embodiments, a PAK inhibitor composition described herein
is
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administered to a patient who has been prescribed an Abeta antibody. Examples
of
antibodies useful in the methods and compositions described herein include and
are not
limited an Abeta antibody (e.g., bapineuzumab), PAK antibodies (e.g.,
ABIN237914) or the
like.
Other Agents
[00284] In some embodiments, one or more PAK inhibitors are used in
combination with
one or more agents that modulate dendritic spine morphology or synaptic
function.
Examples of agents that modulate dendritic spine morphology include
minocycline, trophic
factors (e.g., brain derived neutrophic factor, glial cell-derived neurtrophic
factor), or
anesthetics that modulate spine motility, or the like. In some embodiments,
one or more
PAK inhibitors are used in combination with one or more agents that modulate
cognition. In
some embodiments, a second therapeutic agent is a nootropic agent that
enhances cognition.
Examples of nootropic agents include and are not limited to piracetam,
pramiracetam,
oxiracetam, and aniracetam.
Blood Brain Barrier facilitators
[00285] In some instances, a PAK inhibitor is optionally administered in
combination
with a blood brain barrier facilitator. In certain embodiments, an agent that
facilitates the
transport of a PAK inhibitor is covalently attached to the PAK inhibitor. In
some instances,
PAK inhibitors described herein are modified by covalent attachment to a
lipophilic carrier
or co-formulation with a lipophilic carrier. In some embodiments, a PAK
inhibitor is
covalently attached to a lipophilic carrier, such as e.g., DHA, or a fatty
acid. In some
embodiments, a PAK inhibitor is covalently attached to artificial low density
lipoprotein
particles. In some instances, carrier systems facilitate the passage of PAK
inhibitors
described herein across the blood-brain barrier and include but are not
limited to, the use of
a dihydropyridine pyridinium salt carrier redox system for delivery of drug
species across
the blood brain barrier. In some instances a PAK inhibitor described herein is
coupled to a
lipophilic phosphonate derivative. In certain instances, PAK inhibitors
described herein are
conjugated to PEG-oligomers/polymers or aprotinin derivatives and analogs. In
some
instances, an increase in influx of a PAK inhibitor described herein across
the blood brain
barrier is achieved by modifying A PAK inhibitor described herein (e.g., by
reducing or
increasing the number of charged groups on the compound) and enhancing
affinity for a
blood brain barrier transporter. In certain instances, a PAK inhibitor is co-
administered with
an an agent that reduces or inhibits efflux across the blood brain barrier,
e.g. an inhibitor of
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P-glycoprotein pump (PGP) mediated efflux (e.g., cyclosporin, SCH66336
(lonafarnib,
Schering)).
[00286] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with, e.g., compounds described in U.S. Patents 5,863,532,
6,191,169,
6,248,549, and 6,498,163; U.S. Patent Applications 200200045564, 20020086390,
20020106690,20020142325,20030124107,20030166623,20040091992,20040102623,
20040208880, 200500203114, 20050037965, 20050080002, and 20050233965,
20060088897; EP Patent Publication 1492871; PCT patent publication WO 9902701;
PCT
patent publication WO 2008/047307; Kumar et al., (2006), Nat. Rev. Cancer,
6:459; and
Eswaran et al., (2007), Structure, 15:201-213, all of which are incorporated
herein by
reference for disclosure of kinase inhibitors and/or PAK inhibitors described
therein.
[00287] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with compounds including and not limited to BMS-387032; SNS-
032;
CHI4-258; TKI-258; EKB-569; JNJ-7706621; PKC-412; staurosporine; SU-14813;
sunitinib; N-(3-chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-
ylpropoxy)quinazolin-4-amine (gefitinib), VX-680; MK-0457; combinations
thereof; or
salts, prodrugs thereof
[00288] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a polypeptide comprising an amino acid sequence about 80%
to about
100% identical, e.g., 85%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or any
other
percent from about 80% to about 100% identical the following amino acid
sequence:
HTIHVGFDAVTGEFTGMPEQWARLLQTSNITKSEQKKNPQAVLDVLEFYNSKK
TSNSQ KYMSFTDKS
[00289] The above sequence corresponds to the PAK autoinhibitory domain (PAD)
polypeptide amino acids 83-149 of PAK1 polypeptide as described in, e.g., Zhao
et al
(1998). In some embodiments, the PAK inhibitor is a fusion protein comprising
the above-
described PAD amino acid sequence. In some embodiments, in order to facilitate
cell
penetration the fusion polypeptide (e.g., N-terminal or C-terminal) further
comprises a
polybasic protein transduction domain (PTD) amino acid sequence, e.g.:
RKKRRQRR;
YARAAARQARA; THRLPRRRRRR; or GGRRARRRRRR.
[00290] In some embodiments, in order to enhance uptake into the brain, the
fusion
polypeptide further comprises a human insulin receptor antibody as described
in U.S. Patent
Application Serial No. 11/245,546.
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[00291] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a peptide inhibitor comprising a sequence at least 60% to
100%, e.g.,
65%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or any other
percent from about 60% to about 100% identical the following amino acid
sequence:
PPVIAPREHTKSVYTRS as described in, e.g., Zhao et al (2006), Nat Neurosci,
9(2):234-
242. In some embodiments, the peptide sequence further comprises a PTD amino
acid
sequence as described above.
[00292] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a polypeptide comprising an amino acid sequence at least
80% to
100%, e.g., 85%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or any other percent
from
about 80% to about 100% identical to the FMRP1 protein (GenBank Accession No.
Q06787), where the polypeptide is able to bind with a PAK (for example, PAK1,
PAK2,
PAK3, PAK4, PAKSand/or PAK6). In some embodiments compounds of Formula I-XV
are
optionally administered in combination with a polypeptide comprising an amino
acid
sequence at least 80% to 100%, e.g., 85%, 90%, 92%, 93%, 95%, 96%, 97%, 98%,
99%, or
any other percent from about 80% to about 100% identical to the FMRP 1 protein
(GenBank
Accession No. Q06787), where the polypeptide is able to bind with a Group I
PAK, such as,
for example PAK1 (see, e.g., Hayashi et al (2007), Proc Natl Acad Sci USA,
104(27):11489-11494. In some embodiments, compounds of Formula I-XV are
optionally
administered in combination with a polypeptide comprising a fragment of human
FMRP 1
protein with an amino acid sequence at least 80% to 100%, e.g., 85%, 90%, 92%,
93%,
95%, 96%, 97%, 98%, 99%, or any other percent from about 80% to about 100%
identical
to the sequence of amino acids 207-425 of the human FMRP1 protein (i.e.,
comprising the
KH1 and KH2 domains), where the polypeptide is able to bind to PAK1.
[00293] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a polypeptide comprising an amino acid sequence at least
80% to
100%, e.g., 85%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or any other percent
from
about 80% to about 100% identical to at least five, at least ten at least
twenty, at least thirty,
at least forty, at least fifty, at least sixty, at least seventy, at least
eighty, at least ninety
contiguous amino acids of the huntingtin (htt) protein (GenBank Accession No.
NP 002102,
gi 90903231), where the polypeptide is able to bind to a Group 1 PAK (for
example, PAK1,
PAK2, and/or PAK3). In some embodiments, compounds of Formula I-XV are
optionally
administered in combination with a polypeptide comprising an amino acid
sequence at least
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80% to 100%, e.g., 85%, 90%, 92%, 93%, 95%, 96%, 97%, 98%, 99%, or any other
percent
from about 80% to about 100% identical to at least a portion of the huntingtin
(htt) protein
(GenBank Accession No. NP 002102, gi 90903231), where the polypeptide is able
to bind
to PAK1. In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a polypeptide comprising a fragment of human huntingtin
protein with
an amino acid sequence at least 80% to 100%, e.g., 85%, 90%, 92%, 93%, 95%,
96%, 97%,
98%, 99%, or any other percent from about 80% to about 100% identical to a
sequence of at
least five, at least ten, at least twenty, at least thirty, at least forty, at
least fifty, at least sixty,
at least seventy, at least eighty, at least ninety, or at least 100 contiguous
amino acids of the
human huntingtin protein that is outside of the sequence encoded by exon 1 of
the htt gene
(i.e., a fragment that does not contain poly glutamate domains), where the
polypeptide binds
a PAK. In some embodiments, compounds of Formula I-XV are optionally
administered in
combination with a polypeptide comprising a fragment of human huntingtin
protein with an
amino acid sequence at least 80% identical to a sequence of the human
huntingtin protein
that is outside of the sequence encoded by exon 1 of the htt gene (i.e., a
fragment that does
not contain poly glutamate domains), where the polypeptide binds PAK1.
Upstream regulators of p21 activated kinases
[00294] In certain embodiments, compounds of Formula I-XV are optionally
administered in combination with an indirect PAK modulator (e.g., an indirect
PAK
inhibitor) that affects the activity of a molecule that acts in a signaling
pathway upstream of
PAK (upstream regulators of PAK). Upstream effectors of PAK include, but are
not limited
to: TrkB receptors; NMDA receptors; EphB receptors; adenosine receptors;
estrogen
receptors; integrins; FMRP; Rho-family GTPases, including Cdc42, Rae
(including but not
limited to Racl and Rac2), CDKS, P13 kinases, NCK, PDK1, EKT, GRB2, Chp, TC10,
Tel,
and Wrch- 1; guanine nucleotide exchange factors ("GEFs"), such as but not
limited to
GEFT, members of the Dbl family of GEFs, p21-activated kinase interacting
exchange
factor (PIX), DEF6, Zizimin 1, Vavl, Vav2, Dbs, members of the DOCK180 family,
Kalirin-7, and Tiam1; G protein-coupled receptor kinase-interacting protein 1
(GIT1),
CIB 1, filamin A, Etk/Bmx, and sphingosine.
[00295] Modulators of NMDA receptor include, but are not limited to, 1-
aminoadamantane, dextromethorphan, dextrorphan, ibogaine, ketamine, nitrous
oxide,
phencyclidine, riluzole, tiletamine, memantine, neramexane, dizocilpine,
aptiganel,
remacimide, 7-chlorokynurenate, DCKA (5,7-dichlorokynurenic acid), kynurenic
acid, 1-
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aminocyclopropanecarboxylic acid (ACPC), AP7 (2-amino-7-phosphonoheptanoic
acid),
APV (R-2-amino-5-phosphonopentanoate), CPPene (3-[(R)-2-carboxypiperazin-4-yl]-
prop-
2-enyl-1-phosphonic acid); (+)-(1S, 2S)-1-(4-hydroxy-phenyl)-2-(4-hydroxy-4-
phenylpiperidino)- 1-pro-panol; (1S, 2S)-1-(4-hydroxy-3-methoxyphenyl)-2-(4-
hydroxy-4-
phenylpiperi-dino)-1-propanol; (3R, 45)-3-(4-(4-fluorophenyl)-4-
hydroxypiperidin-l-yl-)-
chroman-4,7-diol; (1R*, 2R*)-1-(4-hydroxy-3-methylphenyl)-2-(4-(4-fluoro-
phenyl)-4-
hydroxypiperidin-l-yl)-propan-l-ol-mesylate; and/or combinations thereof.
[00296] Modulators of estrogen receptors include, and are not limited to, PPT
(4,4',4"-(4-
Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol); SKF-82958 (6-chloro-7,8-
dihydroxy-3-allyl-l-
phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine); estrogen; estradiol; estradiol
derivatives,
including but not limited to 17-(3 estradiol, estrone, estriol, ER(3-131,
phytoestrogen, MK
101 (bioNovo); VG-1010 (bioNovo); DPN (diarylpropiolitrile); ERB-041; WAY-
202196;
WAY-214156; genistein; estrogen; estradiol; estradiol derivatives, including
but not limited
to 17-(3 estradiol, estrone, estriol, benzopyrans and triazolo-
tetrahydrofluorenones, disclosed
in U.S. Patent No. 7,279,499, and Parker et al., Bioorg. & Med. Chem. Ltrs.
16: 4652-4656
(2006), each of which is incorporated herein by reference for such disclosure.
[00297] Modulators of TrkB include by way of example, neutorophic factors
including
BDNF and GDNF. Modulators of EphB include XL647 (Exelixis), EphB modulator
compounds described in WO/2006081418 and US Appl. Pub. No. 20080300245,
incorporated herein by reference for such disclosure, or the like.
[00298] Modulators of integrins include by way of example, ATN-161, PF-
04605412,
MEDI-522, Volociximab, natalizumab, Volociximab, Ro 27-2771, Ro 27-2441,
etaracizumab, CNTO-95, JSM6427, cilengitide, R411 (Roche), EMD 121974,
integrin
antagonist compounds described in J. Med. Chem., 2002, 45 (16), pp 3451-3457,
incorporated herein by reference for such disclosure, or the like.
[00299] Adenosine receptor modulators include, by way of example,
theophylline, 8-
Cyclopentyl- 1,3-dimethylxanthine (CPX), 8-Cyclopentyl-1,3-dipropylxanthine
(DPCPX),
8-Phenyl-1,3-dipropylxanthine, PSB 36, istradefylline, SCH-58261, SCH-442,416,
ZM-
241,385, CVT-6883, MRS-1706, MRS-1754, PSB-603, PSB-0788, PSB-1115, MRS-1191,
MRS-1220, MRS-1334, MRS-1523, MRS-3777, MRE3008F20, PSB-10, PSB-11, VUF-
5574, N6-Cyclopentyladenosine, CCPA, 2'-MeCCPA, GR 79236, SDZ WAG 99, ATL-
146e, CGS-21680, Regadenoson, 5'-N-ethylcarboxamidoadenosine, BAY 60-6583, LUF-
5835, LUF-5 845, 2-(1-Hexynyl)-N-methyladenosine, CF-101 (IB-MECA), 2-Cl-IB-
MECA,
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CP-532,903, MRS-3558, Rosuvastatin, KW-3902, SLV320, mefloquine, regadenoson,
or
the like.
[00300] In some embodiments, compounds reducing PAK levels decrease PAK
transcription or translation or reduce RNA or protein levels. In some
embodiments, a
compound that decreases PAK levels is an upstream effector of PAK. In some
embodiments, exogenous expression of the activated forms of the Rho family
GTPases Chp
and cdc42 in cells leads to increased activation of PAK while at the same time
increasing
turnover of the PAK protein, significantly lowering its level in the cell
(Hubsman et al.
(2007) Biochem. J. 404: 487-497). PAK clearance agents include agents that
increase
expression of one or more Rho family GTPases and/or one or more guanine
nucleotide
exchange factors (GEFs) that regulate the activity of Rho family GTPases, in
which
overexpression of a Rho family GTPase and/or a GEF results in lower levels of
PAK
protein in cells. PAK clearance agents also include agonists of Rho family
GTPases, as well
as agonists of GTP exchange factors that activate Rho family GTPases, such as
but not
limited to agonists of GEFs of the Dbl family that activate Rho family
GTPases.
[00301] Overexpression of a Rho family GTPase is optionally by means of
introducing a
nucleic acid expression construct into the cells or by administering a
compound that induces
transcription of the endogenous gene encoding the GTPase. In some embodiments,
the Rho
family GTPase is Rac (e.g., Rae 1, Rac2, or Rac3), cdc42, Chp, TC10, Tel, or
Wrnch- 1. For
example, a Rho family GTPase includes Racl, Rac2, Rac3, or cdc42. A gene
introduced
into cells that encodes a Rho family GTPase optionally encodes a mutant form
of the gene,
for example, a more active form (for example, a constitutively active form,
Hubsman et al.
(2007) Biochem. J. 404: 487-497). In some embodiments, a PAK clearance agent
is, for
example, a nucleic acid encoding a Rho family GTPase, in which the Rho family
GTPase is
expressed from a constitutive or inducible promoter. PAK levels in some
embodiments are
reduced by a compound that directly or indirectly enhances expression of an
endogenous
gene encoding a Rho family GTPase.
[00302] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a PAK clearance agent.
[00303] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a compound that directly or indirectly decreases the
activation or
activity of the upstream effectors of PAK. For example, in some embodiments a
compound
that inhibits the GTPase activity of the small Rho-family GTPases such as Rac
and cdc42
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thereby reduce the activation of PAK kinase. In some embodiments, the compound
that
decreases PAK activation is by secramine that inhibits cdc42 activation,
binding to
membranes and GTP in the cell (Pelish et al. (2005) Nat. Chem. Biol. 2: 39-
46). In some
embodiments, PAK activation is decreased by EHT 1864, a small molecule that
inhibits
Rae I, Raclb, Rac2 and Rac3 function by preventing binding to guanine
nucleotide
association and engagement with downstream effectors (Shutes et al. (2007) J.
Biol. Chem.
49: 35666-35678). In some embodiments, PAK activation is also decreased by the
NSC23766 small molecule that binds directly to Racl and prevents its
activation by Rac-
specific RhoGEFs (Gao et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101: 7618-
7623). In
some embodiments, PAK activation is also decreased by the 16 kDa fragment of
prolactin
(16k PRL), generated from the cleavage of the 23 kDa prolactin hormone by
matrix
metalloproteases and cathepsin D in various tissues and cell types. 16k PRL
down-regulates
the Ras-Tiaml-Rae l-Pak l signaling pathway by reducing Racl activation in
response to
cell stimuli such as wounding (Lee et al. (2007) Cancer Res 67:11045-11053).
In some
embodiments, PAK activation is decreased by inhibition of NMDA and/or AMPA
receptors. Examples of modulators of AMPA receptors include and are not
limited to
ketamine, MK801, CNQX (6-cyano-7-nitroquinoxaline-2,3-dione); NBQX (2,3-
dihydroxy-
6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione); DNQX (6,7-
dinitroquinoxaline-2,3-
dione); kynurenic acid; 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo-[flquinoxaline;
PCP or the
like. In some embodiments, PAK activation is decreased by inhibition of TrkB
activation. In
some embodiments, PAK activation is decreased by inhibition of BDNF activation
of TrkB.
In some embodiments, compounds of Formula I-XV are optionally administered in
combination with an antibody to BDNF. In some embodiments, PAK activation is
decreased by inhibition of TrkB receptors; NMDA receptors; EphB receptors;
adenosine
receptors; estrogen receptors; integrins; Rho-family GTPases, including Cdc42,
Rae
(including but not limited to Racl and Rac2), CDKS, P13 kinases, NCK, PDK1,
EKT,
GRB2, Chp, TC10, Tel, and Wrch-1; guanine nucleotide exchange factors
("GEFs"), such
as but not limited to GEFT, members of the Dbl family of GEFs, p21-activated
kinase
interacting exchange factor (PIX), DEF6, Zizimin 1, Vavl, Vav2, Dbs, members
of the
DOCK180 family, Kalirin-7, and Tiaml; G protein-coupled receptor kinase-
interacting
protein 1 (GIT1), CIB1, filamin A, Etk/Bmx, and/or binding to FMRP and/or
sphingosine.
[00304] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a compound that decreases PAK levels in the cell, e.g., a
compound
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that directly or indirectly increases the activity of a guanine exchange
factor (GEF) that
promotes the active state of a Rho family GTPase, such as an agonist of a GEF
that
activates a Rho family GTPase, such as but not limited to, Rae or cdc42.
Activation of
GEFs is also effected by compounds that activate TrkB, NMDA, or EphB
receptors.
[00305] In some embodiments, a PAK clearance agent is a nucleic acid encoding
a GEF
that activates a Rho family GTPase, in which the GEF is expressed from a
constitutive or
inducible promoter. In some embodiments, a guanine nucleotide exchange factor
(GEF),
such as but not limited to a GEF that activates a Rho family GTPase is
overexpressed in
cells to increase the activation level of one or more Rho family GTPases and
thereby lower
the level of PAK in cells. GEFs include, for example, members of the Dbl
family of
GTPases, such as but not limited to, GEFT, PIX (e.g., alphaPIX, betaPIX),
DEF6, Zizimin
1, Vav1, Vav2, Dbs, members of the DOCK180 family, hPEM-2, FLJ00018, kalirin,
Tiaml, STEF, DOCK2, DOCK6, DOCK7, DOCKS, Asf, EhGEF3, or GEF-1. In some
embodiments, PAK levels are also reduced by a compound that directly or
indirectly
enhances expression of an endogenous gene encoding a GEF. A GEF expressed from
a
nucleic acid construct introduced into cells is in some embodiments a mutant
GEF, for
example a mutant having enhanced activity with respect to wild type.
[00306] The clearance agent is optionally a bacterial toxin such as Salmonella
typhinmurium toxin SpoE that acts as a GEF to promote cdc42 nucleotide
exchange
(Buchwald et al. (2002) EMBO J. 21: 3286-3295; Schlumberger et al. (2003) J.
Biological
Chem. 278: 27149-27159). Toxins such as SopE, fragments thereof, or peptides
or
polypeptides having an amino acid sequence at least 80% to 100%, e.g., 85%,
90%, 92%,
93%, 95%, 96%, 97%, 98%, 99%, or any other percent from about 80% to about
100%
identical to a sequence of at least five, at least ten, at least twenty, at
least thirty, at least
forty, at least fifty, at least sixty, at least seventy, at least eighty, at
least ninety, or at least
100 contiguous amino acids of the toxin are also optionally used as
downregulators of PAK
activity. The toxin is optionally produced in cells from nucleic acid
constructs introduced
into cells.
Modulators of upstream regulators of PAKs
[00307] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a modulator of an upstream regulator of PAKs. In some
embodiments,
a modulator of an upstream regulator of PAKs is an indirect inhibitor of PAK.
In certain
instances, a modulator of an upstream regulator of PAKs is a modulator of
PDK1. In some
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instances, a modulator of PDK1 reduces of inhibits the activity of PDK1. In
some instances
a PDK1 inhibitor is an antisense compound (e.g., any PDK1 inhibitor described
in U.S.
Patent No. 6,124,272, which PDK1 inhibitor is incorporated herein by
reference). In some
instances, a PDK1 inhibitor is a compound described in e.g., U.S. Patent Nos.
7,344,870,
and 7,041,687, which PDK1 inhibitors are incorporated herein by reference. In
some
embodiments, an indirect inhibitor of PAK is a modulator of a P13 kinase. In
some instances
a modulator of a P13 kinase is a P13 kinase inhibitor. In some instances, a
P13 kinase
inhibitor is an antisense compound (e.g., any P13 kinase inhibitor described
in WO
2001/018023, which P13 kinase inhibitors are incorporated herein by
reference). In some
instances, an inhibitor of a P13 kinase is 3-morpholino-5-phenylnaphthalen-
1(4H)-one
(LY294002), or a peptide based covalent conjugate of LY294002, (e.g., SF 1126,
Semaphore pharmaceuticals). In certain embodiments, an indirect inhibitor of
PAK is a
modulator of Cdc42. In certain embodiments, a modulator of Cdc42 is an
inhibitor of
Cdc42. In certain embodiments, a Cdc42 inhibitor is an antisense compound
(e.g., any
Cdc42 inhibitor described in U.S. Patent No. 6,410,323, which Cdc42 inhibitors
are
incorporated herein by reference). In some instances, an indirect inhibitor of
PAK is a
modulator of GRB2. In some instances, a modulator of GRB2 is an inhibitor of
GRB2. In
some instances a GRB2 inhibitor is a GRb2 inhibitor described in e.g., U.S.
Patent No.
7,229,960, which GRB2 inhibitor is incorporated by reference herein. In
certain
embodiments, an indirect inhibitor of PAK is a modulator of NCK. In certain
embodiments,
an indirect inhibitor of PAK is a modulator of ETK. In some instances, a
modulator of ETK
is an inhibitor of ETK. In some instances an ETK inhibitor is a compound e.g.,
a-Cyano-
(3,5-di-t-butyl-4-hydroxy)thiocinnamide (AG 879).
[00308] In some embodiments, indirect PAK inhibitors act by decreasing
transcription
and/or translation of PAK. An indirect PAK inhibitor in some embodiments
decreases
transcription and/or translation of a PAK. For example, in some embodiments,
modulation
of PAK transcription or translation occurs through the administration of
specific or non-
specific inhibitors of PAK transcription or translation. In some embodiments,
proteins or
non-protein factors that bind the upstream region of the PAK gene or the 5'
UTR of a PAK
mRNA are assayed for their affect on transcription or translation using
transcription and
translation assays (see, for example, Baker, et al. (2003) J Biol. Chem. 278:
17876-17884;
Jiang et al. (2006) J Chromatography A 1133: 83-94; Novoa et al. (1997)
Biochemistry 36:
7802-7809; Brandi et al. (2007) Methods Enzymol. 431: 229-267). PAK inhibitors
include
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DNA or RNA binding proteins or factors that reduce the level of transcription
or translation
or modified versions thereof. In other embodiments, compounds of Formula I-XV
are
optionally administered in combination with an agent that is a modified form
(e.g., mutant
form or chemically modified form) of a protein or other compound that
positively regulates
transcription or translation of PAK, in which the modified form reduces
transcription or
translation of PAK. In yet other embodiments, a transcription or translation
inhibitor is an
antagonist of a protein or compound that positively regulates transcription or
translation of
PAK, or is an agonist of a protein that represses transcription or
translation.
[00309] Regions of a gene other than those upstream of the transcriptional
start site and
regions of an mRNA other than the 5' UTR (such as but not limited to regions
3' of the
gene or in the 3' UTR of an mRNA, or regions within intron sequences of either
a gene or
mRNA) also include sequences to which effectors of transcription, translation,
mRNA
processing, mRNA transport, and mRNA stability bind. In some embodiments,
compounds
of Formula I-XV are optionally administered in combination with a clearance
agent
comprising a polypeptide having homology to an endogenous protein that affects
mRNA
processing, transport, or stability, or is an antagonist or agonist of one or
more proteins that
affect mRNA processing, transport, or turnover, such that the inhibitor
reduces the
expression of PAK protein by interfering with PAK mRNA transport or
processing, or by
reducing the half-life of PAK mRNA. A PAK clearance agents in some embodiments
interferes with transport or processing of a PAK mRNA, or by reducing the half-
life of a
PAK mRNA.
[00310] For example, PAK clearance agents decrease RNA and/or protein half-
life of a
PAK isoform, for example, by directly affecting mRNA and/or protein stability.
In certain
embodiments, PAK clearance agents cause PAK mRNA and/or protein to be more
accessible and/or susceptible to nucleases, proteases, and/or the proteasome.
In some
embodiments, compounds of Formula I-XV are optionally administered in
combination
with agents that decrease the processing of PAK mRNA thereby reducing PAK
activity. For
example, PAK clearance agents function at the level of pre-mRNA splicing, 5'
end
formation (e.g. capping), 3' end processing (e.g. cleavage and/or
polyadenylation), nuclear
export, and/or association with the translational machinery and/or ribosomes
in the
cytoplasm. In some embodiments, PAK clearance agents cause a decrease in the
level of
PAK mRNA and/or protein, the half-life of PAK mRNA and/or protein by at least
about
5%, at least about 10%, at least about 20%, at least about 30%, at least about
40%, at least
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about 50%, at least about 60%, at least about 80%, at least about 90%, at
least about 95%,
or substantially 100%.
[00311] In some embodiments, the clearance agent comprises one or more RNAi or
antisense oligonucleotides directed against one or more PAK isoform RNAs. In
some
embodiments, compounds of Formula I-XV are optionally administered in
combination
with agent that comprise one or more ribozymes directed against one or more
PAK isoform
RNAs. The design, synthesis, and use of RNAi constructs, antisense
oligonucleotides, and
ribozymes are found, for example, in Dykxhoorn et al. (2003) Nat. Rev. Mol.
Cell. Biol. 4:
457-467; Hannon et al. (2004) Nature 431: 371-378; Sarver et al. (1990)
Science 247:1222-
1225; Been et al. (1986) Cell 47:207-216) . In some embodiments, nucleic acid
constructs
that induce triple helical structures are also introduced into cells to
inhibit transcription of
the PAK gene (Helene (1991) Anticancer Drug Des. 6:569-584).
[00312] For example, a clearance agent is in some embodiments an RNAi molecule
or a
nucleic acid construct that produces an RNAi molecule. An RNAi molecule
comprises a
double-stranded RNA of at least about seventeen bases having a 2-3 nucleotide
single-
stranded overhangs on each end of the double-stranded structure, in which one
strand of the
double-stranded RNA is substantially complementary to the target PAK RNA
molecule
whose downregulation is desired. "Substantially complementary" means that one
or more
nucleotides within the double-stranded region are not complementary to the
opposite strand
nucleotide(s). Tolerance of mismatches is optionally assessed for individual
RNAi
structures based on their ability to downregulate the target RNA or protein.
In some
embodiments, RNAi is introduced into the cells as one or more short hairpin
RNAs
("shRNAs") or as one or more DNA constructs that are transcribed to produce
one or more
shRNAs, in which the shRNAs are processed within the cell to produce one or
more RNAi
molecules.
[00313] Nucleic acid constructs for the expression of siRNA, shRNA, antisense
RNA,
ribozymes, or nucleic acids for generating triple helical structures are
optionally introduced
as RNA molecules or as recombinant DNA constructs. DNA constructs for reducing
gene
expression are optionally designed so that the desired RNA molecules are
expressed in the
cell from a promoter that is transcriptionally active in mammalian cells, such
as, for
example, the SV40 promoter, the human cytomegalovirus immediate-early promoter
(CMV
promoter), or the pol III and/or pol II promoter using known methods. For some
purposes, it
is desirable to use viral or plasmid-based nucleic acid constructs. Viral
constructs include
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but are not limited to retroviral constructs, lentiviral constructs, or based
on a pox virus, a
herpes simplex virus, an adenovirus, or an adeno-associated virus (AAV).
[00314] In other embodiments, compounds of Formula I-XV are optionally
administered
in combination with a polypeptide that decreases the activity of PAK. Protein
and peptide
inhibitors of PAK are optionally based on natural substrates of PAK, e.g.,
Myosin light
chain kinase (MLCK), regulatory Myosin light chain (R-MLC), Myosins I heavy
chain,
myosin II heavy chain, Myosin VI, Caldesmon, Desmin, Op18/stathmin, Merlin,
Filamin A,
LIM kinase (LIMK), cortactin, cofilin, Ras, Raf, Mek, p47(phox), BAD, caspase
3, estrogen
and/or progesterone receptors, NET 1, Gaz, phosphoglycerate mutase-B, RhoGDI,
prolactin,
p4lArc, cortactin and/or Aurora-A. In some embodiments, compounds of Formula I-
XV are
optionally administered in combination with an agent that is based on a
sequence of PAK
itself, for example, the autoinhibitory domain in the N-terminal portion of
the PAK protein
that binds the catalytic domain of a partner PAK molecule when the PAK
molecule is in its
homodimeric state (Zhao et al. (1998) Mol. Cell Biol. 18:2153-2163; Knaus et
al. (1998) J
Biol. Chem. 273: 21512-21518; Hofman et al. (2004) J Cell Sci. 117: 4343-
4354). In some
embodiments, polypeptide inhibitors of PAK comprise peptide mimetics, in which
the
peptide has binding characteristics similar to a natural binding partner or
substrate of PAK.
[00315] In some embodiments, provided herein are compounds that downregulate
PAK
protein level. In some embodiments, the compounds described herein activate or
increase
the activity of an upstream regulator or downstream target of PAK. In some
embodiments,
compounds described herein downregulate protein level of a PAK. In some
instances
compounds described herein reduce at least one of the symptoms related a CNS
disorder by
reducing the amount of PAK in a cell. In some embodiments a compound that
decreases
PAK protein levels in cells also decreases the activity of PAK in the cells.
In some
embodiments a compound that decreases PAK protein levels does not have a
substantial
impact on PAK activity in cells. In some embodiments a compound that increases
PAK
activity in cells decreases PAK protein levels in the cells.
[00316] In some embodiments, a compound that decreases the amount of PAK
protein in
cells decreases transcription and/or translation of PAK or increases the
turnover rate of PAK
mRNA or protein by modulating the activity of an upstream effector or
downstream
regulator of PAK. In some embodiments, PAK expression or PAK levels are
influenced by
feedback regulation based on the conformation, chemical modification, binding
status, or
activity of PAK itself In some embodiments, PAK expression or PAK levels are
influenced
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by feedback regulation based on the conformation, chemical modification,
binding status, or
activity of molecules directly or indirectly acted on by PAK signaling
pathways. As used
herein "binding status" refers to any or a combination of whether PAK, an
upstream
regulator of PAK, or a downstream effector of PAK is in a monomeric state or
in an
oligomeric complex with itself, or whether it is bound to other polypeptides
or molecules.
For example, a downstream target of PAK, when phosphorylated by PAK, in some
embodiments directly or indirectly downregulates PAK expression or decrease
the half-life
of PAK mRNA or protein. Downstream targets of PAK include but are not limited
to:
Myosin light chain kinase (MLCK), regulatory Myosin light chain (R-MLC),
Myosins I
heavy chain, myosin II heavy chain, Myosin VI, Caldesmon, Desmin,
Op18/stathmin,
Merlin, Filamin A, LIM kinase (LIMK), Ras, Raf, Mek, p47 ph, BAD, caspase 3,
estrogen
and/or progesterone receptors, NET 1, Gaz, phosphoglycerate mutase-B, RhoGDI,
prolactin,
p41 , cortactin and/or Aurora-A. Downregulators of PAK levels include
downstream
targets of PAK or fragments thereof in a phosphorylated state and downstream
targets of
PAK or fragments thereof in a hyperphosphorylated state.
[00317] A fragment of a downstream target of PAK includes any fragment with an
amino
acid sequence at least 80% to 100%, e.g., 85%, 90%, 92%, 93%, 95%, 96%, 97%,
98%,
99%, or any other percent from about 80% to about 100% identical to a sequence
of at least
five, at least ten, at least twenty, at least thirty, at least forty, at least
fifty, at least sixty, at
least seventy, at least eighty, at least ninety, or at least 100 contiguous
amino acids of the
downstream regulator, in which the fragment of the downstream target of PAK is
able to
downregulate PAK mRNA or protein expression or increase turnover of PAK mRNA
or
protein. In some embodiments, the fragment of a downstream regulator of PAK
comprises a
sequence that includes a phosphorylation site recognized by PAK, in which the
site is
phosphorylated.
[00318] In some embodiments, compounds of Formula I-XV are optionally
administered
in combination with a compound that decreases the level of PAK including a
peptide,
polypeptide, or small molecule that inhibits dephosphorylation of a downstream
target of
PAK, such that phosphorylation of the downstream target remains at a level
that leads to
downregulation of PAK levels.
[00319] In some embodiments, PAK activity is reduced or inhibited via
activation and/or
inhibition of an upstream regulator and/or downstream target of PAK. In some
embodiments, the protein expression of a PAK is downregulated. In some
embodiments, the
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amount of PAK in a cell is decreased. In some embodiments a compound that
decreases
PAK protein levels in cells also decreases the activity of PAK in the cells.
In some
embodiments a compound that decreases PAK protein levels does not decrease PAK
activity in cells. In some embodiments a compound that increases PAK activity
in cells
decreases PAK protein levels in the cells.
[00320] In some instances, compounds of Formula I-XV are optionally
administered in
combination with a polypeptide that is delivered to one or more brain regions
of an
individual by administration of a viral expression vector, e.g., an AAV
vector, a lentiviral
vector, an adenoviral vector, or a HSV vector. A number of viral vectors for
delivery of
therapeutic proteins are described in, e.g., U.S. Patent Nos., 7,244,423,
6,780,409,
5,661,033. In some embodiments, the PAK inhibitor polypeptide to be expressed
is under
the control of an inducible promoter (e.g., a promoter containing a tet-
operator). Inducible
viral expression vectors include, for example, those described in U.S. Patent
No. 6,953,575.
Inducible expression of a PAK inhibitor polypeptide allows for tightly
controlled and
reversible increases of PAK inhibitor polypeptide expression by varying the
dose of an
inducing agent (e.g., tetracycline) administered to an individual.
Anti-cancer Agents
[00321] Where the subject is suffering from or at risk of suffering from a B-
cell
proliferative disorder (e.g., plasma cell myeloma), the subjected in some
embodiments is
treated with a compound of Formula I-XV in any combination with one or more
other anti-
cancer agents. In some embodiments, one or more of the anti-cancer agents are
proapoptotic agents. Examples of anti-cancer agents include, but are not
limited to, any of
the following: gossyphol, genasense, polyphenol E, Chlorofusin, all trans-
retinoic acid
(ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL), 5-
aza-2'-deoxycytidine, all trans retinoic acid, doxorubicin, vincristine,
etoposide,
gemcitabine, imatinib (Gleevec ), geldanamycin, 17-N-Allylamino-17-
Demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib,
trastuzumab,
BAY 11-7082, PKC412, or PD 184352, TaxolTM, also referred to as "paclitaxel",
which is
an anti-cancer drug which acts by enhancing and stabilizing microtubule
formation, and
analogs of Taxo1TM, such as TaxotereTM. Compounds that have the basic taxane
skeleton as
a common structure feature, have also been shown to have the ability to arrest
cells in the
G2-M phases due to stabilized microtubules and in some embodiments are useful
for
treating cancer in combination with the compounds described herein.
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[00322] Further examples of anti-cancer agents for use in combination with a
compound
of Formula I-XV include inhibitors of mitogen-activated protein kinase
signaling, e.g.,
U0126, PD98059, PD184352, PD0325901, ARRY-142886, SB239063, SP600125, BAY
43-9006, wortmannin, or LY294002; Syk inhibitors; mTOR inhibitors; and
antibodies (e.g.,
rituxan).
[00323] Other anti-cancer agents that can be employed in combination with an
irreversible Btk inhibitor compound include Adriamycin, Dactinomycin,
Bleomycin,
Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin;
aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide;
amsacrine;
anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa;
azotomycin;
batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide
dimesylate;
bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan;
cactinomycin;
calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride;
carzelesin; cedefingol; chlorambucil; cirolemycin; cladribine; crisnatol
mesylate;
cyclophosphamide; cytarabine; dacarbazine; daunorubicin hydrochloride;
decitabine;
dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; doxorubicin;
doxorubicin
hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin;
edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate;
epipropidine;
epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine;
estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine;
fadrozole
hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate;
fluorouracil;
flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride;
hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II
(including
recombinant interleukin II, or rIL2), interferon alfa-2a; interferon alfa-2b;
interferon alfa-
n 1; interferon alfa-n3; interferon beta-1 a; interferon gamma-1 b;
iproplatin; irinotecan
hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole
hydrochloride;
lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol;
maytansine;
mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate;
melphalan;
menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine;
meturedepa;
mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin;
mitosper;
mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole;
nogalamycin;
ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine; peplomycin
sulfate;
perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin;
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plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride;
puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide;
safingol;
safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin;
spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin;
streptozocin;
sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride;
temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa;
tiazofurin;
tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate;
trimetrexate;
trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil
mustard; uredepa;
vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;
vindesine sulfate;
vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine
tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin;
zorubicin
hydrochloride.
[00324] Other anti-cancer agents that in some embodiments are employed in
combination
with a compound of Formula I-XV include: 20-epi-1, 25 dihydroxyvitamin D3; 5-
ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin;
aldesleukin;
ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid;
amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis
inhibitors;
antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-
1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense
oligonucleotides;
aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators;
apurinic acid; ara-
CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1;
axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III
derivatives;
balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;
beta
lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A;
bizelesin; breflate;
bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin
derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole;
carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;
carzelesin;
casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorlns;
chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene
analogues;
clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin
analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A
derivatives;
curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine
ocfosfate;
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cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B;
deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin
B;
didox; diethylnorspermine; dihydro-5-azacytidine; 9-dioxamycin; diphenyl
spiromustine;
docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA;
ebselen;
ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur;
epirubicin;
epristeride; estramustine analogue; estrogen agonists; estrogen antagonists;
etanidazole;
etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide;
filgrastim; fmasteride;
flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin
hydrochloride;
forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin;
gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione
inhibitors;
hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;
idarubicin;
idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor;
interferon
agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-
; iroplact;
irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F;
lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan
sulfate; leptolstatin;
letrozole; leukemia inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear
polyamine
analogue; lipophilic disaccharide peptide; lipophilic platinum compounds;
lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin;
loxoribine;
lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine;
mannostatin A;
marimastat; masoprocol; maspin; matrilysin inhibitors; matrix
metalloproteinase inhibitors;
menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor;
mifepristone; miltefosine; mirimostim; mismatched double stranded RNA;
mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth
factor-saporin;
mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic
gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol;
multiple
drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy;
mustard
anticancer agent; mycaperoxide B; mycobacterial cell wall extract;
myriaporone; N-
acetyldinaline; N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine;
napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral
endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide
antioxidant;
nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides;
onapristone;
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ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin;
osaterone; oxaliplatin;
oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol;
panomifene;
parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate
sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin;
phenylacetate;
phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin;
piritrexim; placetin
A; placetin B; plasminogen activator inhibitor; platinum complex; platinum
compounds;
platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl
bis-
acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune
modulator;
protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein
tyrosine
phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins;
pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylerie conjugate; raf
antagonists;
raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras
inhibitors; ras-GAP
inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes;
R<sub>11</sub> retinamide; rogletimide; rohitukine; romurtide; roquinimex;
rubiginone B 1;
ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1
mimetics;
semustine; senescence derived 1; sense oligonucleotides; signal transduction
inhibitors;
signal transduction modulators; single chain antigen-binding protein;
sizofuran;
sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin
binding
protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1;
squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide;
stromelysin
inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist;
suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen
methiodide;
tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium;
telomerase inhibitors;
temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine;
thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin;
thymopoietin receptor
agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin;
tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell factor;
translation
inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate;
triptorelin; tropisetron;
turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors;
ubenimex; urogenital
sinus-derived growth inhibitory factor; urokinase receptor antagonists;
vapreotide; variolin
B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin;
vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;
and zinostatin
stimalamer.
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[00325] Yet other anticancer agents that in further embodiments are employed
in
combination with a compound of Formula I-XV include alkylating agents,
antimetabolites,
natural products, or hormones, e.g., nitrogen mustards (e.g.,
mechloroethamine,
cyclophosphamide, chlorambucil, etc.), alkyl sulfonates (e.g., busulfan),
nitrosoureas (e.g.,
carmustine, lomusitne, etc.), or triazenes (decarbazine, etc.). Examples of
antimetabolites
include but are not limited to folic acid analog (e.g., methotrexate), or
pyrimidine analogs
(e.g., Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine,
pentostatin).
[00326] Examples of natural products useful in combination with a compound of
Formula I-XV include but are not limited to vinca alkaloids (e.g., vinblastin,
vincristine),
epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin,
doxorubicin,
bleomycin), enzymes (e.g., L-asparaginase), or biological response modifiers
(e.g.,
interferon a).
[00327] Examples of alkylating agents that in further embodiments are employed
in
combination with a compound of Formula I-XV include, but are not limited to,
nitrogen
mustards (e.g., mechloroethamine, cyclophosphamide, chlorambucil, melphalan,
etc.),
ethylenimine and methylmelamines (e.g., hexamethlymelamine, thiotepa), alkyl
sulfonates
(e.g., busulfan), nitrosoureas (e.g., carmustine, lomusitne, semustine,
streptozocin, etc.), or
triazenes (decarbazine, etc.). Examples of antimetabolites include, but are
not limited to
folic acid analog (e.g., methotrexate), or pyrimidine analogs (e.g.,
fluorouracil, floxuridine,
Cytarabine), purine analogs (e.g., mercaptopurine, thioguanine, pentostatin.
[00328] Examples of hormones and antagonists useful in combination with a
compound
of Formula I-XV include, but are not limited to, adrenocorticosteroids (e.g.,
prednisone),
progestins (e.g., hydroxyprogesterone caproate, megestrol acetate,
medroxyprogesterone
acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol),
antiestrogen (e.g., tamoxifen),
androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogen
(e.g., flutamide),
gonadotropin releasing hormone analog (e.g., leuprolide). Other agents that
can be used in
the methods and compositions described herein for the treatment or prevention
of cancer
include platinum coordination complexes (e.g., cisplatin, carboblatin),
anthracenedione
(e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), methyl hydrazine
derivative (e.g.,
procarbazine), adrenocortical suppressant (e.g., mitotane, aminoglutethimide).
[00329] Examples of anti-cancer agents which act by arresting cells in the G2-
M phases
due to stabilized microtubules and which in other embodiments are used in
combination
with a compound of Formula I-XV include without limitation the following
marketed drugs
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and drugs in development: Erbulozole (also known as R-55104), Dolastatin 10
(also known
as DLS-10 and NSC-376128), Mivobulin isethionate (also known as CI-980),
Vincristine,
NSC-639829, Discodermolide (also known as NVP-XX-A-296), ABT-751 (Abbott, also
known as E-7010), Altorhyrtins (such as Altorhyrtin A and Altorhyrtin C),
Spongistatins
(such as Spongistatin 1, Spongistatin 2, Spongistatin 3, Spongistatin 4,
Spongistatin 5,
Spongistatin 6, Spongistatin 7, Spongistatin 8, and Spongistatin 9), Cemadotin
hydrochloride (also known as LU-103793 and NSC-D-669356), Epothilones (such as
Epothilone A, Epothilone B, Epothilone C (also known as desoxyepothilone A or
dEpoA),
Epothilone D (also referred to as KOS-862, dEpoB, and desoxyepothilone B),
Epothilone E,
Epothilone F, Epothilone B N-oxide, Epothilone A N-oxide, 16-aza-epothilone B,
21-
aminoepothilone B (also known as BMS-3 10705), 21-hydroxyepothilone D (also
known as
Desoxyepothilone F and dEpoF), 26-fluoroepothilone), Auristatin PE (also known
as NSC-
654663), Soblidotin (also known as TZT-1027), LS-4559-P (Pharmacia, also known
as LS-
4577), LS-4578 (Pharmacia, also known as LS-477-P), LS-4477 (Pharmacia), LS-
4559
(Pharmacia), RPR-112378 (Aventis), Vincristine sulfate, DZ-3358 (Daiichi), FR-
182877
(Fujisawa, also known as WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2
(Hungarian Academy of Sciences), BSF-223651 (BASF, also known as ILX-651 and
LU-
223651), SAH-49960 (Lilly/Novartis), SDZ-268970 (Lilly/Novartis), AM-97
(Armad/Kyowa Hakko), AM- 132 (Armad), AM-138 (Armad/Kyowa Hakko), IDN-5005
(Indena), Cryptophycin 52 (also known as LY-355703), AC-7739 (Ajinomoto, also
known
as AVE-8063A and CS-39.HC1), AC-7700 (Ajinomoto, also known as AVE-8062, AVE-
8062A, CS-39-L-Ser.HC1, and RPR-258062A), Vitilevuamide, Tubulysin A,
Canadensol,
Centaureidin (also known as NSC-106969), T-138067 (Tularik, also known as T-
67, TL-
138067 and TI-138067), COBRA-1 (Parker Hughes Institute, also known as DDE-261
and
WHI-261), H10 (Kansas State University), H16 (Kansas State University),
Oncocidin Al
(also known as BTO-956 and DIME), DDE-313 (Parker Hughes Institute),
Fijianolide B.
Laulimalide, SPA-2 (Parker Hughes Institute), SPA-1 (Parker Hughes Institute,
also known
as SPIKET-P), 3-IAABU (Cytoskeleton/Mt. Sinai School of Medicine, also known
as MF-
569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-
105972
(Abbott), Hemiasterlin, 3-BAABU (Cytoskeleton/Mt. Sinai School of Medicine,
also
known as MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate,
T-
138026 (Tularik), Monsatrol, Inanocine (also known as NSC-698666), 3-1AABE
(Cytoskeleton/Mt. Sinai School of Medicine), A-204197 (Abbott), T-607
(Tuiarik, also
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known as T-900607), RPR-115781 (Aventis), Eleutherobins (such as
Desmethyleleutherobin, Desaetyleleutherobin, Isoeleutherobin A, and Z-
Eleutherobin),
Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144
(Asta
Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A,
TUB-
245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistin (also known
as NSCL-
96F037), D-68838 (Asta Medica), D-68836 (Asta Medica), Myoseverin B, D-43411
(Zentaris, also known as D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-
286 (also
known as SPA-110, trifluoroacetate salt) (Wyeth), D-82317 (Zentaris), D-82318
(Zentaris),
SC-12983 (NCI), Resverastatin phosphate sodium, BPR-OY-007 (National Health
Research
Institutes), and SSR-250411 (Sanofi).
[00330] Any combination of one or more PAK inhibitors and a second therapeutic
agent
is compatible with any method described herein. The PAK inhibitor compositions
described
herein are also optionally used in combination with other therapeutic reagents
that are
selected for their therapeutic value for the condition to be treated. In
general, the
compositions described herein and, in embodiments where combinational therapy
is
employed, other agents do not have to be administered in the same
pharmaceutical
composition, and, because of different physical and chemical characteristics,
are optionally
administered by different routes. The initial administration is generally made
according to
established protocols, and then, based upon the observed effects, the dosage,
modes of
administration and times of administration subsequently modified.
[00331] In certain instances, it is appropriate to administer at least one PAK
inhibitor
composition described herein in combination with another therapeutic agent. By
way of
example only, if one of the side effects experienced by a patient upon
receiving one of the
PAK inhibitor compositions described herein is nausea, then it is appropriate
to administer
an anti-nausea agent in combination with the initial therapeutic agent. Or, by
way of
example only, the therapeutic effectiveness of a PAK inhibitor is enhanced by
administration of an adjuvant (i.e., by itself the adjuvant has minimal
therapeutic benefit,
but in combination with another therapeutic agent, the overall therapeutic
benefit to the
patient is enhanced). Or, by way of example only, the benefit experienced by a
patient is
increased by administering a PAK inhibitor with another therapeutic agent
(which also
includes a therapeutic regimen) that also has therapeutic benefit. In any
case, regardless of
the disease, disorder or condition being treated, the overall benefit
experienced by the
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patient is either simply additive of the two therapeutic agents or the patient
experiences a
synergistic benefit.
[00332] Therapeutically-effective dosages vary when the drugs are used in
treatment
combinations. Suitable methods for experimentally determining therapeutically-
effective
dosages of drugs and other agents include, e.g., the use of metronomic dosing,
i.e.,
providing more frequent, lower doses in order to minimize toxic side effects.
Combination
treatment further includes periodic treatments that start and stop at various
times to assist
with the clinical management of the patient.
[00333] In any case, the multiple therapeutic agents (one of which is a PAK
inhibitor
described herein) is administered in any order, or even simultaneously. If
simultaneously,
the multiple therapeutic agents are optionally provided in a single, unified
form, or in
multiple forms (by way of example only, either as a single pill or as two
separate pills). In
some embodiments, one of the therapeutic agents is given in multiple doses, or
both are
given as multiple doses. If not simultaneous, the timing between the multiple
doses
optionally varies from more than zero weeks to less than four weeks. In
addition, the
combination methods, compositions and formulations are not to be limited to
the use of
only two agents; the use of multiple therapeutic combinations are also
envisioned.
[00334] The pharmaceutical agents which make up the combination therapy
disclosed
herein are optionally a combined dosage form or in separate dosage forms
intended for
substantially simultaneous administration. The pharmaceutical agents that make
up the
combination therapy are optionally also be administered sequentially, with
either
therapeutic compound being administered by a regimen calling for two-step
administration.
The two-step administration regimen optionally calls for sequential
administration of the
active agents or spaced-apart administration of the separate active agents.
The time period
between the multiple administration steps ranges from, a few minutes to
several hours,
depending upon the properties of each pharmaceutical agent, such as potency,
solubility,
bioavailability, plasma half-life and kinetic profile of the pharmaceutical
agent. Circadian
variation of the target molecule concentration are optionally used to
determine the optimal
dose interval.
[00335] In addition, a PAK inhibitor is optionally used in combination with
procedures
that provide additional or synergistic benefit to the patient. By way of
example only,
patients are expected to find therapeutic and/or prophylactic benefit in the
methods
described herein, wherein pharmaceutical composition of a PAK inhibitor and
/or
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combinations with other therapeutics are combined with genetic testing to
determine
whether that individual is a carrier of a mutant gene that is correlated with
certain diseases
or conditions.
[00336] A PAK inhibitor and the additional therapy(ies) are optionally
administered
before, during or after the occurrence of a disease or condition, and the
timing of
administering the composition containing a PAK inhibitor varies in some
embodiments.
Thus, for example, the PAK inhibitor is used as a prophylactic and
administered
continuously to individuals with a propensity to develop conditions or
diseases in order to
prevent the occurrence of the disease or condition. The PAK inhibitors and
compositions
are optionally administered to an individual during or as soon as possible
after the onset of
the symptoms. The administration of the compounds are optionally initiated
within the first
48 hours of the onset of the symptoms, preferably within the first 48 hours of
the onset of
the symptoms, more preferably within the first 6 hours of the onset of the
symptoms, and
most preferably within 3 hours of the onset of the symptoms. The initial
administration is
optionally via any route practical, such as, for example, an intravenous
injection, a bolus
injection, infusion over 5 minutes to about 5 hours, a pill, a capsule,
transdermal patch,
buccal delivery, and the like, or combination thereof. A PAK inhibitor is
optionally
administered as soon as is practicable after the onset of a disease or
condition is detected or
suspected, and for a length of time necessary for the treatment of the
disease, such as, for
example, from about 1 month to about 3 months. The length of treatment
optionally varies
for each individual, and the length is then determined using the known
criteria. For
example, the PAK inhibitor or a formulation containing the PAK inhibitor is
administered
for at least 2 weeks, preferably about 1 month to about 5 years, and more
preferably from
about 1 month to about 3 years.
[00337] In some embodiments, the particular choice of compounds depends upon
the
diagnosis of the attending physicians and their judgment of the condition of
an individual
and the appropriate treatment protocol. The compounds are optionally
administered
concurrently (e.g., simultaneously, essentially simultaneously or within the
same treatment
protocol) or sequentially, depending upon the nature of the disease, disorder,
or condition,
the condition of an individual, and the actual choice of compounds used. In
certain
instances, the determination of the order of administration, and the number of
repetitions of
administration of each therapeutic agent during a treatment protocol, is based
on an
evaluation of the disease being treated and the condition of an individual.
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[00338] In some embodiments, therapeutically-effective dosages vary when the
drugs are
used in treatment combinations. Methods for experimentally determining
therapeutically-
effective dosages of drugs and other agents for use in combination treatment
regimens are
described in the literature.
[00339] In some embodiments of the combination therapies described herein,
dosages of
the co-administered compounds vary depending on the type of co-drug employed,
on the
specific drug employed, on the disease or condition being treated and so
forth. In addition,
when co-administered with one or more biologically active agents, the compound
provided
herein is optionally administered either simultaneously with the biologically
active agent(s),
or sequentially. In certain instances, if administered sequentially, the
attending physician
will decide on the appropriate sequence of therapeutic compound described
herein in
combination with the additional therapeutic agent.
[00340] The multiple therapeutic agents (at least one of which is a
therapeutic compound
described herein) are optionally administered in any order or even
simultaneously. If
simultaneously, the multiple therapeutic agents are optionally provided in a
single, unified
form, or in multiple forms (by way of example only, either as a single pill or
as two separate
pills). In certain instances, one of the therapeutic agents is optionally
given in multiple
doses. In other instances, both are optionally given as multiple doses. If not
simultaneous,
the timing between the multiple doses is any suitable timing, e.g, from more
than zero
weeks to less than four weeks. In some embodiments, the additional therapeutic
agent is
utilized to achieve reversal or amelioration of symptoms of a CNS disorder,
whereupon the
therapeutic agent described herein (e.g., a compound of any one of Formula I-
XV is
subsequently administered. In addition, the combination methods, compositions
and
formulations are not to be limited to the use of only two agents; the use of
multiple
therapeutic combinations are also envisioned (including two or more compounds
described
herein).
[00341] In certain embodiments, a dosage regimen to treat, prevent, or
ameliorate the
condition(s) for which relief is sought, is modified in accordance with a
variety of factors.
These factors include the disorder from which an individual suffers, as well
as the age,
weight, sex, diet, and medical condition of an individual. Thus, in various
embodiments, the
dosage regimen actually employed varies and deviates from the dosage regimens
set forth
herein.
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Examples of Pharmaceutical Compositions and Methods of Administration
[00342] Provided herein, in certain embodiments, are compositions comprising a
therapeutically effective amount of any compound described herein (e.g., a
compound of
Formula I-XV.
[00343] Pharmaceutical compositions are formulated using one or more
physiologically
acceptable carriers including excipients and auxiliaries which facilitate
processing of the
active compounds into preparations which are used pharmaceutically. Proper
formulation is
dependent upon the route of administration chosen. A summary of pharmaceutical
compositions is found, for example, in Remington: The Science and Practice of
Pharmacy,
Nineteenth Ed (Ea hston, Pa.: Mack Publishing Company, 1995); Hoover, John E.,
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania
1975;
Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel
Decker,
New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery
Systems,
Seventh Ed. (Lippincott Williams & Wilkins, 1999).
[00344] Provided herein are pharmaceutical compositions that include one or
more PAK
inhibitors and a pharmaceutically acceptable diluent(s), excipient(s), or
carrier(s). In
addition, the PAK inhibitor is optionally administered as pharmaceutical
compositions in
which it is mixed with other active ingredients, as in combination therapy. In
some
embodiments, the pharmaceutical compositions includes other medicinal or
pharmaceutical
agents, carriers, adjuvants, such as preserving, stabilizing, wetting or
emulsifying agents,
solution promoters, salts for regulating the osmotic pressure, and/or buffers.
In addition, the
pharmaceutical compositions also contain other therapeutically valuable
substances.
[00345] A pharmaceutical composition, as used herein, refers to a mixture of a
PAK
inhibitor with other chemical components, such as carriers, stabilizers,
diluents, dispersing
agents, suspending agents, thickening agents, and/or excipients. The
pharmaceutical
composition facilitates administration of the PAK inhibitor to an organism. In
practicing the
methods of treatment or use provided herein, therapeutically effective amounts
of a PAK
inhibitor are administered in a pharmaceutical composition to a mammal having
a condition,
disease, or disorder to be treated. Preferably, the mammal is a human. A
therapeutically
effective amount varies depending on the severity and stage of the condition,
the age and
relative health of an individual, the potency of the PAK inhibitor used and
other factors. The
PAK inhibitor is optionally used singly or in combination with one or more
therapeutic
agents as components of mixtures.
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[00346] The pharmaceutical formulations described herein are optionally
administered to
an individual by multiple administration routes, including but not limited to,
oral, parenteral
(e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical,
rectal, or
transdermal administration routes. By way of example only, Example 26a is
describes a
parenteral formulation, Example 26f describes a rectal formulation. The
pharmaceutical
formulations described herein include, but are not limited to, aqueous liquid
dispersions,
self-emulsifying dispersions, solid solutions, liposomal dispersions,
aerosols, solid dosage
forms, powders, immediate release formulations, controlled release
formulations, fast melt
formulations, tablets, capsules, pills, delayed release formulations, extended
release
formulations, pulsatile release formulations, multiparticulate formulations,
and mixed
immediate and controlled release formulations.
[00347] The pharmaceutical compositions will include at least one PAK
inhibitor, as an
active ingredient in free-acid or free-base form, or in a pharmaceutically
acceptable salt
form. In addition, the methods and pharmaceutical compositions described
herein include
the use of N-oxides, crystalline forms (also known as polymorphs), as well as
active
metabolites of these PAK inhibitors having the same type of activity. In some
situations,
PAK inhibitors exist as tautomers. All tautomers are included within the scope
of the
compounds presented herein. Additionally, the PAK inhibitor exists in
unsolvated as well as
solvated forms with pharmaceutically acceptable solvents such as water,
ethanol, and the
like. The solvated forms of the PAK inhibitors presented herein are also
considered to be
disclosed herein.
[00348] "Carrier materials" include any commonly used excipients in
pharmaceutics and
should be selected on the basis of compatibility with compounds disclosed
herein, such as, a
PAK inhibitor, and the release profile properties of the desired dosage form.
Exemplary
carrier materials include, e.g., binders, suspending agents, disintegration
agents, filling
agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents,
diluents, and the like.
[00349] Moreover, the pharmaceutical compositions described herein, which
include a
PAK inhibitor, are formulated into any suitable dosage form, including but not
limited to,
aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries,
suspensions and the like, for
oral ingestion by a patient to be treated, solid oral dosage forms, aerosols,
controlled release
formulations, fast melt formulations, effervescent formulations, lyophilized
formulations,
tablets, powders, pills, dragees, capsules, delayed release formulations,
extended release
formulations, pulsatile release formulations, multiparticulate formulations,
and mixed
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immediate release and controlled release formulations. In some embodiments, a
formulation
comprising a PAK inhibitor is a solid drug dispersion. A solid dispersion is a
dispersion of
one or more active ingredients in an inert carrier or matrix at solid state
prepared by the
melting (or fusion), solvent, or melting-solvent methods. (Chiou and
Riegelman, Journal of
Pharmaceutical Sciences, 60, 1281 (1971)). The dispersion of one or more
active agents in a
solid diluent is achieved without mechanical mixing. Solid dispersions are
also called solid-
state dispersions. In some embodiments, any compound described herein (e.g., a
compound
of Formula I-XV is formulated as a spray dried dispersion (SDD). An SDD is a
single phase
amorphous molecular dispersion of a drug in a polymer matrix. It is a solid
solution
prepared by dissolving the drug and a polymer in a solvent (e.g., acetone,
methanol or the
like) and spray drying the solution. The solvent rapidly evaporates from
droplets which
rapidly solidifies the polymer and drug mixture trapping the drug in amorphous
form as an
amorphous molecular dispersion. In some embodiments, such amorphous
dispersions are
filled in capsules and/or constituted into oral powders for reconstitution.
Solubility of an
SDD comprising a drug is higher than the solubility of a crystalline form of a
drug or a non-
SDD amorphous form of a drug. In some embodiments of the methods described
herein,
PAK inhibitors are administered as SDDs constituted into appropriate dosage
forms
described herein.
[00350] Pharmaceutical preparations for oral use are optionally obtained by
mixing one
or more solid excipient with a PAK inhibitor, optionally grinding the
resulting mixture, and
processing the mixture of granules, after adding suitable auxiliaries, if
desired, to obtain
tablets or dragee cores. Suitable excipients include, for example, fillers
such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such
as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth,
methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose,
sodium
carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or
povidone) or
calcium phosphate. If desired, disintegrating agents are added, such as the
cross linked
croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt
thereof such as
sodium alginate.
[00351] Dragee cores are provided with suitable coatings. For this purpose,
concentrated
sugar solutions are generally used, which optionally contain gum arabic, talc,
polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium
dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments are
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optionally added to the tablets or dragee coatings for identification or to
characterize
different combinations of active compound doses.
[00352] In some embodiments, the solid dosage forms disclosed herein are in
the form of
a tablet, (including a suspension tablet, a fast-melt tablet, a bite-
disintegration tablet, a
rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a
powder (including a
sterile packaged powder, a dispensable powder, or an effervescent powder) a
capsule
(including both soft or hard capsules, e.g., capsules made from animal-derived
gelatin or
plant-derived HPMC, or "sprinkle capsules"), solid dispersion, solid solution,
bioerodible
dosage form, controlled release formulations, pulsatile release dosage forms,
multiparticulate dosage forms, pellets, granules, or an aerosol. By way of
example, Example
26b describes a solid dosage formulation that is a capsule. In other
embodiments, the
pharmaceutical formulation is in the form of a powder. In still other
embodiments, the
pharmaceutical formulation is in the form of a tablet, including but not
limited to, a fast-
melt tablet. Additionally, pharmaceutical formulations of a PAK inhibitor are
optionally
administered as a single capsule or in multiple capsule dosage form. In some
embodiments,
the pharmaceutical formulation is administered in two, or three, or four,
capsules or tablets.
[00353] In another aspect, dosage forms include microencapsulated
formulations. In
some embodiments, one or more other compatible materials are present in the
microencapsulation material. Exemplary materials include, but are not limited
to, pH
modifiers, erosion facilitators, anti-foaming agents, antioxidants, flavoring
agents, and
carrier materials such as binders, suspending agents, disintegration agents,
filling agents,
surfactants, solubilizers, stabilizers, lubricants, wetting agents, and
diluents.
[00354] Exemplary microencapsulation materials useful for delaying the release
of the
formulations including a PAK inhibitor, include, but are not limited to,
hydroxypropyl
cellulose ethers (HPC) such as Klucel or Nisso HPC, low-substituted
hydroxypropyl
cellulose ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such as
Seppifilm-LC, Pharmacoat , Metolose SR, Methocel -E, Opadry YS, PrimaFlo,
Benecel
MP824, and Benecel MP843, methylcellulose polymers such as Methocel -A,
hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS) and
Metolose , Ethylcelluloses (EC) and mixtures thereof such as E461, Ethocel ,
Aqualon -
EC, Surelease , Polyvinyl alcohol (PVA) such as Opadry AMB,
hydroxyethylcelluloses
such as Natrosol , carboxymethylcelluloses and salts of
carboxymethylcelluloses (CMC)
such as Aqualon -CMC, polyvinyl alcohol and polyethylene glycol co-polymers
such as
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Kollicoat IR , monoglycerides (Myverol), triglycerides (KLX), polyethylene
glycols,
modified food starch, acrylic polymers and mixtures of acrylic polymers with
cellulose
ethers such as Eudragit EPO, Eudragit L30D-55, Eudragit FS 30D Eudragit
L100-
55, Eudragit L100, Eudragit S100, Eudragit RD100, Eudragit E100, Eudragit
L12.5, Eudragit S12.5, Eudragit NE30D, and Eudragit NE 40D, cellulose
acetate
phthalate, sepifilms such as mixtures of HPMC and stearic acid, cyclodextrins,
and mixtures
of these materials.
[00355] The pharmaceutical solid oral dosage forms including formulations
described
herein, which include a PAK inhibitor, are optionally further formulated to
provide a
controlled release of the PAK inhibitor. Controlled release refers to the
release of the PAK
inhibitor from a dosage form in which it is incorporated according to a
desired profile over
an extended period of time. Controlled release profiles include, for example,
sustained
release, prolonged release, pulsatile release, and delayed release profiles.
In contrast to
immediate release compositions, controlled release compositions allow delivery
of an agent
to an individual over an extended period of time according to a predetermined
profile. Such
release rates provide therapeutically effective levels of agent for an
extended period of time
and thereby provide a longer period of pharmacologic response while minimizing
side
effects as compared to conventional rapid release dosage forms. Such longer
periods of
response provide for many inherent benefits that are not achieved with the
corresponding
short acting, immediate release preparations.
[00356] In other embodiments, the formulations described herein, which include
a PAK
inhibitor, are delivered using a pulsatile dosage form. A pulsatile dosage
form is capable of
providing one or more immediate release pulses at predetermined time points
after a
controlled lag time or at specific sites. Pulsatile dosage forms including the
formulations
described herein, which include a PAK inhibitor, are optionally administered
using a variety
of pulsatile formulations that include, but are not limited to, those
described in U.S. Pat.
Nos. 5,011,692, 5,017,381, 5,229,135, and 5,840,329. Other pulsatile release
dosage forms
suitable for use with the present formulations include, but are not limited
to, for example,
U.S. Pat. Nos. 4,871,549, 5,260,068, 5,260,069, 5,508,040, 5,567,441 and
5,837,284.
[00357] Liquid formulation dosage forms for oral administration are optionally
aqueous
suspensions selected from the group including, but not limited to,
pharmaceutically
acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and
syrups. See,
e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp.
754-757
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(2002). In addition to the PAK inhibitor, the liquid dosage forms optionally
include
additives, such as: (a) disintegrating agents; (b) dispersing agents; (c)
wetting agents; (d) at
least one preservative, (e) viscosity enhancing agents, (f) at least one
sweetening agent, and
(g) at least one flavoring agent. In some embodiments, the aqueous dispersions
further
includes a crystal-forming inhibitor.
[00358] In some embodiments, the pharmaceutical formulations described herein
are
self-emulsifying drug delivery systems (SEDDS). Emulsions are dispersions of
one
immiscible phase in another, usually in the form of droplets. Generally,
emulsions are
created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or
microemulsions, spontaneously form emulsions when added to an excess of water
without
any external mechanical dispersion or agitation. An advantage of SEDDS is that
only gentle
mixing is required to distribute the droplets throughout the solution.
Additionally, water or
the aqueous phase is optionally added just prior to administration, which
ensures stability of
an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an
effective
delivery system for oral and parenteral delivery of hydrophobic active
ingredients. In some
embodiments, SEDDS provides improvements in the bioavailability of hydrophobic
active
ingredients. Methods of producing self-emulsifying dosage forms include, but
are not
limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563.
[00359] Suitable intranasal formulations include those described in, for
example, U.S.
Pat. Nos. 4,476,116, 5,116,817 and 6,391,452. Nasal dosage forms generally
contain large
amounts of water in addition to the active ingredient. Minor amounts of other
ingredients
such as pH adjusters, emulsifiers or dispersing agents, preservatives,
surfactants, gelling
agents, or buffering and other stabilizing and solubilizing agents are
optionally present.
[00360] For administration by inhalation, the PAK inhibitor is optionally in a
form as an
aerosol, a mist or a powder. Pharmaceutical compositions described herein are
conveniently
delivered in the form of an aerosol spray presentation from pressurized packs
or a nebuliser,
with the use of a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a pressurized
aerosol, the dosage unit is determined by providing a valve to deliver a
metered amount.
Capsules and cartridges of, such as, by way of example only, gelatin for use
in an inhaler or
insufflator are formulated containing a powder mix of the PAK inhibitor and a
suitable
powder base such as lactose or starch. By way of example, Example 26e
describes an
inhalation formulation.
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[00361] Buccal formulations that include a PAK inhibitor include, but are not
limited to,
U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136. In addition,
the buccal
dosage forms described herein optionally further include a bioerodible
(hydrolysable)
polymeric carrier that also serves to adhere the dosage form to the buccal
mucosa. The
buccal dosage form is fabricated so as to erode gradually over a predetermined
time period,
wherein the delivery of the PAK inhibitor, is provided essentially throughout.
Buccal drug
delivery avoids the disadvantages encountered with oral drug administration,
e.g., slow
absorption, degradation of the active agent by fluids present in the
gastrointestinal tract
and/or first-pass inactivation in the liver. The bioerodible (hydrolysable)
polymeric carrier
generally comprises hydrophilic (water-soluble and water-swellable) polymers
that adhere
to the wet surface of the buccal mucosa. Examples of polymeric carriers useful
herein
include acrylic acid polymers and co, e.g., those known as "carbomers"
(Carbopol , which
may be obtained from B.F. Goodrich, is one such polymer). Other components
also be
incorporated into the buccal dosage forms described herein include, but are
not limited to,
disintegrants, diluents, binders, lubricants, flavoring, colorants,
preservatives, and the like.
For buccal or sublingual administration, the compositions optionally take the
form of
tablets, lozenges, or gels formulated in a conventional manner. By way of
example,
Examples 26c and 26d describe sublingual formulations.
[00362] Transdermal formulations of a PAK inhibitor are administered for
example by
those described in U.S. Pat. Nos. 3,598,122, 3,598,123, 3,710,795, 3,731,683,
3,742,951,
3,814,097, 3,921,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894,
4,060,084,
4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168,
5,665,378,
5,837,280, 5,869,090, 6,923,983, 6,929,801 and 6,946,144. By way of example,
Example
26g describes a topical formulation.
[00363] The transdermal formulations described herein include at least three
components: (1) a formulation of a PAK inhibitor; (2) a penetration enhancer;
and (3) an
aqueous adjuvant. In addition, transdermal formulations include components
such as, but
not limited to, gelling agents, creams and ointment bases, and the like. In
some
embodiments, the transdermal formulation further includes a woven or non-woven
backing
material to enhance absorption and prevent the removal of the transdermal
formulation from
the skin. In other embodiments, the transdermal formulations described herein
maintain a
saturated or supersaturated state to promote diffusion into the skin.
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[00364] In some embodiments, formulations suitable for transdermal
administration of a
PAK inhibitor employ transdermal delivery devices and transdermal delivery
patches and
are lipophilic emulsions or buffered, aqueous solutions, dissolved and/or
dispersed in a
polymer or an adhesive. Such patches are optionally constructed for
continuous, pulsatile, or
on demand delivery of pharmaceutical agents. Still further, transdermal
delivery of the PAK
inhibitor is optionally accomplished by means of iontophoretic patches and the
like.
Additionally, transdermal patches provide controlled delivery of the PAK
inhibitor. The rate
of absorption is optionally slowed by using rate-controlling membranes or by
trapping the
PAK inhibitor within a polymer matrix or gel. Conversely, absorption enhancers
are used to
increase absorption. An absorption enhancer or carrier includes absorbable
pharmaceutically acceptable solvents to assist passage through the skin. For
example,
transdermal devices are in the form of a bandage comprising a backing member,
a reservoir
containing the PAK inhibitor optionally with carriers, optionally a rate
controlling barrier to
deliver the PAK inhibitor to the skin of the host at a controlled and
predetermined rate over
a prolonged period of time, and means to secure the device to the skin.
[00365] Formulations that include a PAK inhibitor suitable for intramuscular,
subcutaneous, or intravenous injection include physiologically acceptable
sterile aqueous or
non-aqueous solutions, dispersions, suspensions or emulsions, and sterile
powders for
reconstitution into sterile injectable solutions or dispersions. Examples of
suitable aqueous
and non-aqueous carriers, diluents, solvents, or vehicles including water,
ethanol, polyols
(propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like),
suitable mixtures
thereof, vegetable oils (such as olive oil) and injectable organic esters such
as ethyl oleate.
Proper fluidity is maintained, for example, by the use of a coating such as
lecithin, by the
maintenance of the required particle size in the case of dispersions, and by
the use of
surfactants. Formulations suitable for subcutaneous injection also contain
optional additives
such as preserving, wetting, emulsifying, and dispensing agents.
[00366] For intravenous injections, a PAK inhibitor is optionally formulated
in aqueous
solutions, preferably in physiologically compatible buffers such as Hank's
solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration,
penetrants appropriate to the barrier to be permeated are used in the
formulation. For other
parenteral injections, appropriate formulations include aqueous or nonaqueous
solutions,
preferably with physiologically compatible buffers or excipients.
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[00367] Parenteral injections optionally involve bolus injection or continuous
infusion.
Formulations for injection are optionally presented in unit dosage form, e.g.,
in ampoules or
in multi dose containers, with an added preservative. In some embodiments, the
pharmaceutical composition described herein are in a form suitable for
parenteral injection
as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles,
and contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical
formulations for parenteral administration include aqueous solutions of the
PAK inhibitor in
water soluble form. Additionally, suspensions of the PAK inhibitor are
optionally prepared
as appropriate oily injection suspensions.
[00368] In some embodiments, the PAK inhibitor is administered topically and
formulated into a variety of topically administrable compositions, such as
solutions,
suspensions, lotions, gels, pastes, medicated sticks, balms, creams or
ointments. Such
pharmaceutical compositions optionally contain solubilizers, stabilizers,
tonicity enhancing
agents, buffers and preservatives.
[00369] The PAK inhibitor is also optionally formulated in rectal compositions
such as
enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly
suppositories, or
retention enemas, containing conventional suppository bases such as cocoa
butter or other
glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG,
and the like.
In suppository forms of the compositions, a low-melting wax such as, but not
limited to, a
mixture of fatty acid glycerides, optionally in combination with cocoa butter
is first melted.
Examples of Methods of Dosing and Treatment Regimens
[00370] The PAK inhibitor is optionally used in the preparation of medicaments
for the
prophylactic and/or therapeutic treatment of a CNS disorder that would
benefit, at least in
part, from amelioration of symptoms. In addition, a method for treating any of
the diseases
or conditions described herein in an individual in need of such treatment,
involves
administration of pharmaceutical compositions containing at least one PAK
inhibitor
described herein, or a pharmaceutically acceptable salt, pharmaceutically
acceptable N-
oxide, pharmaceutically active metabolite, pharmaceutically acceptable
prodrug, or
pharmaceutically acceptable solvate thereof, in therapeutically effective
amounts to said
individual.
[00371] In the case wherein the patient's condition does not improve, upon the
doctor's
discretion the administration of the PAK inhibitor is optionally administered
chronically,
that is, for an extended period of time, including throughout the duration of
the patient's life
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in order to ameliorate or otherwise control or limit the symptoms of the
patient's disease or
condition.
[00372] In the case wherein the patient's status does improve, upon the
doctor's
discretion the administration of the PAK inhibitor is optionally given
continuously;
alternatively, the dose of drug being administered is temporarily reduced or
temporarily
suspended for a certain length of time (i.e., a "drug holiday"). The length of
the drug
holiday optionally varies between 2 days and 1 year, including by way of
example only, 2
days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20
days, 28 days, 35
days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250
days, 280
days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a
drug holiday
includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,
30%,
35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%,95%, or 100%.
[00373] Once improvement of the patient's conditions has occurred, a
maintenance dose
is administered if necessary. Subsequently, the dosage or the frequency of
administration, or
both, is reduced, as a function of the symptoms, to a level at which the
improved disease,
disorder or condition is retained. In some embodiments, patients require
intermittent
treatment on a long-term basis upon any recurrence of symptoms.
[00374] In some embodiments, the pharmaceutical compositions described herein
are in
unit dosage forms suitable for single administration of precise dosages. In
unit dosage form,
the formulation is divided into unit doses containing appropriate quantities
of one or more
PAK inhibitor. In some embodiments, the unit dosage is in the form of a
package containing
discrete quantities of the formulation. Non-limiting examples are packaged
tablets or
capsules, and powders in vials or ampoules. In some embodiments, aqueous
suspension
compositions are packaged in single-dose non-reclosable containers.
Alternatively,
multiple-dose reclosable containers are used, in which case it is typical to
include a
preservative in the composition. By way of example only, formulations for
parenteral
injection are presented in unit dosage form, which include, but are not
limited to ampoules,
or in multi dose containers, with an added preservative.
[00375] The daily dosages appropriate for the PAK inhibitor are from about
0.01 to about
2.5 mg/kg per body weight. An indicated daily dosage in the larger mammal,
including, but
not limited to, humans, is in the range from about 0.5 mg to about 1000 mg,
conveniently
administered in divided doses, including, but not limited to, up to four times
a day or in
extended release form. Suitable unit dosage forms for oral administration
include from
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about 1 to about 500 mg active ingredient, from about 1 to about 250 mg of
active
ingredient, or from about 1 to about 100 mg active ingredient. The foregoing
ranges are
merely suggestive, as the number of variables in regard to an individual
treatment regime is
large, and considerable excursions from these recommended values are not
uncommon.
Such dosages are optionally altered depending on a number of variables, not
limited to the
activity of the PAK inhibitor used, the disease or condition to be treated,
the mode of
administration, the requirements of an individual, the severity of the disease
or condition
being treated, and the judgment of the practitioner.
[00376] Toxicity and therapeutic efficacy of such therapeutic regimens are
optionally
determined in cell cultures or experimental animals, including, but not
limited to, the
determination of the LD50 (the dose lethal to 50% of the population) and the
ED50 (the
dose therapeutically effective in 50% of the population). The dose ratio
between the toxic
and therapeutic effects is the therapeutic index, which is expressed as the
ratio between
LD50 and ED50. PAK inhibitors exhibiting high therapeutic indices are
preferred. The data
obtained from cell culture assays and animal studies is optionally used in
formulating a
range of dosage for use in human. The dosage of such PAK inhibitors lies
preferably within
a range of circulating concentrations that include the ED50 with minimal
toxicity. The
dosage optionally varies within this range depending upon the dosage form
employed and
the route of administration utilized.
Assays for identification and characterization of PAK inhibitors
[00377] Small molecule PAK inhibitors are optionally identified in high-
throughput in
vitro or cellular assays as described in, e.g., Yu et al (2001), JBiochem
(Tokyo);
129(2):243-25 1; Rininsland et al (2005), BMC Biotechnol, 5:16; and Allen et
al (2006),
ACS Chem Biol; 1(6):371-376. PAK inhibitors suitable for the methods described
herein are
available from a variety of sources including both natural (e.g., plant
extracts) and synthetic.
For example, candidate PAK inhibitors are isolated from a combinatorial
library, i.e., a
collection of diverse chemical compounds generated by either chemical
synthesis or
biological synthesis by combining a number of chemical "building blocks." For
example, a
linear combinatorial chemical library such as a polypeptide library is formed
by combining
a set of chemical building blocks called amino acids in every possible way for
a given
compound length (i.e., the number of amino acids in a polypeptide compound).
Millions of
chemical compounds can be synthesized through such combinatorial mixing of
chemical
building blocks, as desired. Theoretically, the systematic, combinatorial
mixing of 100
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interchangeable chemical building blocks results in the synthesis of 100
million tetrameric
compounds or 10 billion pentameric compounds. See Gallop et al. (1994), J.
Med. Chem.
37(9), 1233. Each member of a library may be singular and/or may be part of a
mixture (e.g.
a "compressed library"). The library may comprise purified compounds and/or
may be
"dirty" (i.e., containing a quantity of impurities). Preparation and screening
of combinatorial
chemical libraries are documented methodologies. See Cabilly, ed., Methods in
Molecular
Biology, Humana Press, Totowa, NJ, (1998). Combinatorial chemical libraries
include, but
are not limited to: diversomers such as hydantoins, benzodiazepines, and
dipeptides, as
described in, e.g., Hobbs et al. (1993), Proc. Natl. Acad. Sci. U.S.A. 90,
6909; analogous
organic syntheses of small compound libraries, as described in Chen et al.
(1994), J. Amer.
Chem. Soc., 116: 2661; Oligocarbamates, as described in Cho, et al. (1993),
Science 261,
1303; peptidyl phosphonates, as described in Campbell et al. (1994), J. Org.
Chem., 59:
658; and small organic molecule libraries containing, e.g., thiazolidinones
and
metathiazanones (U.S. Pat. No. 5,549,974), pyrrolidines (U.S. Pat. Nos.
5,525,735 and
5,519,134), benzodiazepines (U.S. Pat. No. 5,288,514). In addition, numerous
combinatorial
libraries are commercially available from, e.g., ComGenex (Princeton, NJ);
Asinex
(Moscow, Russia); Tripos, Inc. (St. Louis, MO); ChemStar, Ltd. (Moscow,
Russia); 3D
Pharmaceuticals (Exton, PA); and Martek Biosciences (Columbia, MD).
[00378] Devices for the preparation of combinatorial libraries are
commercially available
(see, e.g., 357 MPS, 390 MPS from Advanced Chem Tech, Louisville, KY; Symphony
from
Rainin, Woburn, MA; 433A from Applied Biosystems, Foster City, CA; and 9050
Plus
from Millipore, Bedford, MA). A number of robotic systems have also been
developed for
solution phase chemistries. These systems include automated workstations like
the
automated synthesis apparatus developed by Takeda Chemical Industries, LTD
(Osaka,
Japan), and many robotic systems utilizing robotic arms (Zymate II). Any of
the above
devices are optionally used to generate combinatorial libraries for
identification and
characterization of PAK inhibitors which mimic the manual synthetic operations
performed
by small molecule PAK inhibitors suitable for the methods described herein.
Any of the
above devices are optionally used to identify and characterize small molecule
PAK
inhibitors suitable for the methods disclosed herein. In many of the
embodiments disclosed
herein, PAK inhibitors, PAK binding molecules, and PAK clearance agents are
disclosed as
polypeptides or proteins (where polypeptides comprise two or more amino
acids). In these
embodiments, the inventors also contemplate that PAK inhibitors, binding
molecules, and
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clearance agents also include peptide mimetics based on the polypeptides, in
which the
peptide mimetics interact with PAK or its upstream or downstream regulators by
replicating
the binding or substrate interaction properties of PAK or its regulators.
Nucleic acid
aptamers are also contemplated as PAK inhibitors, binding molecules, and
clearance agents,
as are small molecules other than peptides or nucleic acids. For example, in
some
embodiments small molecule PAK binding partners, inhibitors, or clearance
agents, or small
molecule agonists or antagonists of PAK modulators or targets, are designed or
selected
based on analysis of the structure of PAK or its modulators or targets and
binding
interactions with interacting molecules, using "rational drug design" (see,
for example
Jacobsen et al. (2004) Molecular Interventions 4:337-347; Shi et al. (2007)
Bioorg. Med.
Chem. Lett. 17:6744-6749).
[00379] The identification of potential PAK inhibitors is determined by, for
example,
assaying the in vitro kinase activity of PAK in the presence of candidate
inhibitors. In such
assays, PAK and/or a characteristic PAK fragment produced by recombinant means
is
contacted with a substrate in the presence of a phosphate donor (e.g., ATP)
containing
radiolabeled phosphate, and PAK-dependent incorporation is measured.
"Substrate"
includes any substance containing a suitable hydroxyl moiety that can accept
the 7-
phosphate group from a donor molecule such as ATP in a reaction catalyzed by
PAK. The
substrate may be an endogenous substrate of PAK, i.e. a naturally occurring
substance that
is phosphorylated in unmodified cells by naturally-occurring PAK or any other
substance
that is not normally phosphorylated by PAK in physiological conditions, but
may be
phosphorylated in the employed conditions. The substrate may be a protein or a
peptide, and
the phosphrylation reaction may occur on a serine and/or threonine residue of
the substrate.
For example, specific substrates, which are commonly employed in such assays
include, but
are not limited to, histone proteins and myelin basic protein. In some
embodiments, PAK
inhibitors are identified using IMAP technology.
[00380] Detection of PAK dependent phosphorylation of a substrate can be
quantified by
a number of means other than measurement of radiolabeled phosphate
incorporation. For
example, incorporation of phosphate groups may affect physiochemical
properties of the
substrate such as electrophoretic mobility, chromatographic properties, light
absorbance,
fluorescence, and phosphorescence. Alternatively, monoclonal or polyclonal
antibodies can
be generated which selectively recognize phosphorylated forms of the substrate
from non-
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phosphorylated forms whereby allowing antibodies to function as an indicator
of PAK
kinase activity.
[00381] High-throughput PAK kinase assays can be performed in, for example,
microtiter plates with each well containing PAK kinase or an active fragment
thereof,
substrate covalently linked to each well, P32 radiolabled ATP and a potential
PAK inhibitor
candidate. Microtiter plates can contain 96 wells or 1536 wells for large
scale screening of
combinatorial library compounds. After the phosphorylation reaction has
completed, the
plates are washed leaving the bound substrate. The plates are then detected
for phosphate
group incorporation via autoradiography or antibody detection. Candidate PAK
inhibitors
are identified by their ability to decease the amount of PAK
phosphotransferase ability upon
a substrate in comparison with PAK phosphotransferase ability alone.
[00382] The identification of potential PAK inhibitors may also be determined,
for
example, via in vitro competitive binding assays on the catalytic sites of PAK
such as the
ATP binding site and/or the substrate binding site. For binding assays on the
ATP binding
site, a known protein kinase inhibitor with high affinity to the ATP binding
site is used such
as staurosporine. Staurosporine is immobilized and may be fluorescently
labeled,
radiolabeled or in any manner that allows detection. The labeled staurosporine
is introduced
to recombinantly expressed PAK protein or a fragment thereof along with
potential PAK
inhibitor candidates. The candidate is tested for its ability to compete, in a
concentration-
dependant manner, with the immobilized staurosporine for binding to the PAK
protein. The
amount of staurosporine bound PAK is inversely proportional to the affinity of
the
candidate inhibitor for PAK. Potential inhibitors would decrease the
quantifiable binding of
staurosporine to PAK. See e.g., Fabian et al (2005) Nat. Biotech., 23:329.
Candidates
identified from this competitive binding assay for the ATP binding site for
PAK would then
be further screened for selectivity against other kinases for PAK specificity.
[00383] The identification of potential PAK inhibitors may also be determined,
for
example, by in cyto assays of PAK activity in the presence of the inhibitor
candidate.
Various cell lines and tissues may be used, including cells specifically
engineered for this
purpose. In cyto screening of inhibitor candidates may assay PAK activity by
monitoring
the downstream effects of PAK activity. Such effects include, but are not
limited to, the
formation of peripheral actin microspikes and or associated loss of stress
fibers as well as
other cellular responses such as growth, growth arrest, differentiation, or
apoptosis. See e.g.,
Zhao et al., (1998) Mol. Cell. Biol. 18:2153. For example in a PAK yeast
assay, yeast cells
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grow normally in glucose medium. Upon exposure to galactose however,
intracellular PAK
expression is induced, and in turn, the yeast cells die. Candidate compounds
that inhibit
PAK activity are identified by their ability to prevent the yeast cells from
dying from PAK
activation.
[00384] Alternatively, PAK-mediated phosphorylation of a downstream target of
PAK
can be observed in cell based assays by first treating various cell lines or
tissues with PAK
inhibitor candidates followed by lysis of the cells and detection of PAK
mediated events.
Cell lines used in this experiment may include cells specifically engineered
for this purpose.
PAK mediated events include, but are not limited to, PAK mediated
phosphorylation of
downstream PAK mediators. For example, phosphorylation of downstream PAK
mediators
can be detected using antibodies that specifically recognize the
phosphorylated PAK
mediator but not the unphosphorylated form. These antibodies have been
described in the
literature and have been extensively used in kinase screening campaigns. In
some instances
a phospho LIMK antibody is used after treatment of HeLa cells stimulated with
EGF or
sphingosine to detect downstream PAK signaling events.
[00385] The identification of potential PAK inhibitors may also be determined,
for
example, by in vivo assays involving the use of animal models, including
transgenic animals
that have been engineered to have specific defects or carry markers that can
be used to
measure the ability of a candidate substance to reach and/or affect different
cells within the
organism. For example, DISCI knockout mice have defects in synaptic plasticity
and
behavior from increased numbers of dendritic spines and an abundance of long
and
immature spines. Thus, identification of PAK inhibitors can comprise
administering a
candidate to DISCI knockout mice and observing for reversals in synaptic
plasticity and
behavior defects as a readout for PAK inhibition.
[00386] For example, fragile X mental retardation 1 (FMR1) knockout mice have
defects
in synaptic plasticity and behavior from increased numbers of dendritic spines
and an
abundance of long and immature spines. See e.g., Comery et al., (1997) Proc.
Natl. Acad.
Sci. USA, 94:5401-04. As PAK is a downstream effector of the FMR1 gene, the
defects are
reversed upon the use of dominant negative transgenes of PAK that inhibit
endogenous
PAK activity. See Hayashi et al. (2007) Proc. Natl. Acad. Sci. USA, 104:11489-
94. Thus,
identification of PAK inhibitors can comprise administering a candidate to
FMR1 knockout
mice and observing for reversals in synaptic plasticity and behavior defects
as a readout for
PAK inhibition.
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[00387] For example, suitable animal models for Alzheimer's disease are knock-
ins or
transgenes of the human mutated genes including transgenes of the "swedish"
mutation of
APP (APPswe), transgenes expressing the mutant form of presenilin 1 and
presenilin 2
found in familial/early onset AD. Thus, identification of PAK inhibitors can
comprise
administering a candidate to a knock-in animal and observing for reversals in
synaptic
plasticity and behavior defects as a readout for PAK inhibition.
[00388] Administration of the candidate to the animal is via any clinical or
non-clinical
route, including but not limited to oral, nasal, buccal and/or topical
administrations.
Additionally or alternatively, administration may be intratracheal
instillation, bronchial
instillation, intradermal, subcutaneous, intramuscular, intraperitoneal,
inhalation, and/or
intravenous injection.
[00389] Changes in spine morphology are detected using any suitable method,
e.g., by
use of 3D and/or 4D real time interactive imaging and visualization. In some
instances, the
Imaris suite of products (available from Bitplane Scientific Solutions)
provides functionality
for visualization, segmentation and interpretation of 3D and 4D microscopy
datasets
obtained from confocal and wide field microscopy data.
EXAMPLES
[00390] The following specific examples are to be construed as merely
illustrative, and
not limitative of the remainder of the disclosure in any way whatsoever.
[00391] All synthetic chemistry was performed in standard laboratory glassware
unless
indicated otherwise in the examples. Commercial reagents were used as
received. 1H NMR
was performed on a Bruker DRX-400 at 400 MHz. Microwave reactions were
performed in
a Biotage Initiator using the instrument software to control heating time and
pressure.
Hydrogenation reactions were performed on a H-Cube using the commercially
available
catalyst cartridges. Silica gel chromatography was performed manually or on an
ISCO
system with gradient elution as needed.
[00392] Analytical LC/MS method A was performed on an Agilent 1200 system with
a
variable wavelength detector and Agilent 6140 Single quadrupole mass
spectrometer,
alternating positive and negative ion scans. Retention times were determined
from the
extracted 220 nm chromatogram.
HPLC column: Kinetex, 2.6 m, C18, 50 x 2.1mm, maintained at 40 C.
HPLC Gradient: 1.0 mL/min, 95:5:0.1 water:acetonitrile:formic acid to 5:95:0.1
water:acetonitrile:formic acid in 2.5 min, maintaining for 0.5 min.
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[00393] Analytical LC/MS method B was performed on a Shimadzu system with an
attached API 165 single quadrupole mass spectrometer. Retention times were
determined
from the 220 nm chromatogram.
HPLC column: Phenomenex, C18, 2.5 m, 20 x 2mm, maintained at 25 C.
HPLC Gradient: 0.5 mL/min, 95:5:0.02 water: acetonitrile:CF3OOOH to 5:95:0.02
water: acetonitrile:CF3OOOH in 2.9 min, maintaining for 0.9 min.
[00394] Preparative HPLC method A: Preparative HPLC was performed on a Waters
1525/2487 with UV detection at 220 nm and manual collection.
HPLC column: Zorbax SB-C18 21.2 x 100 mm.
HPLC Gradient: 20 mL/min, 95:5:0.1 water: methanol: formic acid to 5:95:0.1
water: methanol: formic acid; the gradient shape was optimized for individual
separations.
[00395] Preparative HPLC method B:
HPLC column: Reprosil-Pur C18-AQ 250 x 20 mm.
HPLC Gradient: 25 mL/min, 25:75:0.02 acetonitrile:water:trifluoroacetic acid
to 100:0:0.02
acetonitrile:water:trifluoroacetic acid; the gradient shape was optimized for
individual
separations.
Example 1: Synthesis of 6-(2-chloro-4-[1,3,4] oxadiazol-2-yl-phenyl)-8-ethyl-2-
[4-(4-
methyl-piperazin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidin-7-one (8).
[00396] Preparation of Intermediate compounds:
[00397] Intermediate 1: Synthesis of 6-bromo-8-ethyl-2-(methylthio)pyrido[2,3-
d]pyrimidin-7(8H)-one (3).
Br Br
1 2 N N O
S N N O S J N N 0
3
[00398] Step 1: Synthesis of 6-bromo-2-(methylthio)pyrido[2,3-d]pyrimidin-
7(8H)-
one (2).
[00399] To a solution of 2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (1,
1.00 g,
5.18 mmol) in anhydrous dimethylformamide (25 mL) was added N-bromosuccinimide
(0.99 g, 5.59 mmol) portionwise at room temperature, and the reaction mixture
was stirred
for 18 h. The mixture was concentrated, and the solid was triturated with hot
water (1 x 20
mL), filtered, and washed with isopropanol to give title compound as a pale
yellow solid
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(0.68 g, 2.50 mmol, 48%). ESMS m/z 272 (M+H)+; iH NMR (400 MHz, DMSO-d6) 6 ppm
12.88 (br. s., 1H), 8.84 (s, 1H), 8.47 (s, 1H), 2.57 (s, 3H).
[00400] Step 2: Synthesis of 6-bromo-8-ethyl-2-(methylthio)pyrido[2,3-
d]pyrimidin-
7(8H)-one (3).
[00401] To a suspension of NaH (60%, 0.15 g, 3.75 mmol) in anhydrous
dimethylformamide (10 mL) was added 6-bromo-2-(methylthio)pyrido[2,3-
d]pyrimidin-
7(8H)-one (2, 0.68 g, 2.50 mmol) at room temperature and the reaction was
stirred at 50 C
for 0.5 h. The reaction mixture was cooled to room temperature, ethyl bromide
(0.22 mL,
0.32 g, 2.93 mmol) was added, and the reaction was stirred at 50 C for 1.5 h.
The mixture
was poured into ice water (10 g), and the white precipitate was collected to
give 6-bromo-8-
ethyl-2-(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (3, 0.57 g, 1.90 mmol,
76%). ESMS
m/z 300 (M+H)+. The material was used without any further purification.
[00402] Synthesis of 6-(2-chloro-4-[1,3,4]oxadiazol-2-phenyl)-8-ethyl-2-[4-(4-
methyl-piperazin-l-yl)-phenylamino]-8H-Ryrido[2,3-d]pyrimidin-7-one (8).
B B N")
r r N i \ \ Br
S N N O N N` O \/^!
3 0 4 \ H N N O
5
N O/ N N NH2
ON / \ \ \ N / \ \ \
\ I NN N O CI \ I NN N O CI
H 6 H 7
N-
N O
N
ON N \ \
i
NN N O CI
H
8
[00403] Step 3: Synthesis of 6-bromo-8-ethyl-2-methanesulfinyl-8H-pyrido[2,3-
d]pyrimidin-7-one (4).
[00404] To a solution of 6-bromo-8-ethyl-2-methylsulfanyl-8H-pyrido[2,3-
d]pyrimidin-
7-one (3, 0.96 g, 3.19 mmol) in dichloromethane (40 mL) was added 3-
chloroperbenzoic
acid (77 %, 0.68 g, 3.04 mmol) in dichloromethane (10 mL) at 0-5 C and the
mixture was
stirred at 0-5 C for lh. The reaction mixture was washed with 10% sodium
bicarbonate
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solution (1 x 20 mL) and water (1 x 20 mL). The organic layer was dried over
sodium
sulfate, filtered and evaporated. The title compound was obtained as a pale
yellow solid
(0.98 g, 3.10 mmol, 97 %). ESMS m/z 316 (M+H)+.
[00405] Step 4: Synthesis of 6-bromo-8-ethyl-2-[4-(4-methyl-piperazin-1-yl)-
phenylamino]-8H-pyrido[2,3-d]pyrimidin-7-one (5).
[00406] 6-Bromo-8-ethyl-2-methanesulfinyl-8H-pyrido[2,3-d]pyrimidin-7-one (4,
600
mg, 1.90 mmol) and 4-(4-methylpiperazino)aniline (363 mg, 1.90 mmol) were
stirred at 120
C for 3 h. The reaction mixture was purified by column chromatography using
dichloromethane:methanol (100:3-*100:5) to give the title compound (340 mg,
0.77 mmol,
40 %) as a yellow solid. ESMS m/z 443 (M+H)+; 1H NMR (400 MHz, CDC13) 6 ppm
8.47
(s, 1H) 7.92 (s, 1H) 7.51 (d, J= 8.8 Hz, 2H) 7.24 (br. s., 1H) 6.96 (d, J= 8.8
Hz, 2H) 4.48
(q, J= 7.0 Hz, 2H) 3.13 - 3.29 (m, 4H) 2.53 - 2.64 (m, 4H) 2.36 (s, 3H) 1.35
(t, J= 7.0 Hz,
3H).
[00407] Step 5: Synthesis of 3-chloro-4-{8-ethyl-2-[4-(4-methyl-piperazin-1-
yl)-
phenylamino]-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-6-yl}benzoic acid methyl
ester
(6).
[00408] 6-Bromo-8-ethyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-8H-
pyrido[2,3-
d]pyrimidin-7-one (5, 110 mg, 0.25 mmol), 2-chloro-4-(methoxycarbonyl)benzene
boronic
acid (58 mg, 0.27 mmol), K3PO4 (58 mg, 0.27 mmol) and PdC12(dppf) (20 mg, 0.02
mmol)
were mixed under argon in a degassed mixture of dimethylformamide and water
(20:1, 4.5
mL). The resulting suspension was irradiated for 30 min at 140 C in a
microwave reactor.
The reaction mixture was evaporated and the residue was purified by column
chromatography, eluting with dichloromethane:methanol (95:5). The title
compound (78
mg, 0.15 mmol, 60%) was obtained as a yellow solid. ESMS m/z 533 (M+H)+; 1H
NMR
(400 MHz, CDC13) 6 ppm 8.55 (s, 1H) 8.15 (d, J= 1.5 Hz, 1H) 7.97 (dd, J= 7.9,
1.5 Hz,
1H) 7.51 - 7.64 (m, 3H) 7.48 (d, J= 7.8 Hz, 1H) 7.27 (br. s., 1H) 6.97 (d, J=
9.0 Hz, 2H)
4.49 (q, J= 7.3 Hz, 2H) 3.95 (s, 3H) 3.18 - 3.35 (m, 4H) 2.67 (br. s., 4H)
2.42 (br. s., 3H)
1.38 (t, J= 7.3 Hz, 3H).
[00409] Step 6: Synthesis of 3-chloro-4-{8-ethyl-2-[4-(4-methyl-piperazin-1-
yl)-
phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidin-6-yl}-benzoic acid
hydrazide
(7).
[00410] 3-Chloro-4-{8-ethyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-
7,8-
dihydro-pyrido[2,3-d]pyrimidin-6-yl}benzoic acid methyl ester (6, 77 mg, 0.14
mmol) in
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the mixture of ethanol (4 mL) and hydrazine hydrate (1 mL) was heated at
reflux for 2 h.
The reaction mixture was cooled and the yellow precipitate was collected and
washed with
2-propanol and diethyl ether to afford the title compound (40 mg, 0.08 mmol,
57 %) as a
yellow solid. ESMS m/z 533 (M+H)+; 1H NMR (400 MHz, DMSO-d6) 6 ppm 9.94 (br.
s.,
2H) 8.77 (s, 1H) 7.95 (d, J= 1.5 Hz, 1H) 7.87 (s, 1H) 7.83 (dd, J= 7.8, 1.5
Hz, 1H) 7.66 (d,
J=9.0Hz,2H)7.51(d,J=7.8Hz,1H)6.94(d,J=9.0Hz,2H)4.56(br.s.,2H)4.36(q,J
= 7.0 Hz, 2H) 3.05 - 3.15 (m, 4H) 2.42 - 2.48 (m, 4H) 2.22 (s, 3H) 1.28 (t, J=
7.0 Hz, 3H).
[00411] Step 7: Synthesis of 6-(2-chloro-4-[1,3,4]oxadiazol-2-yl-phenyl)-8-
ethyl-2-[4-
(4-methyl-piperazin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidin-7-one (8).
[00412] 3-Chloro-4-{8-ethyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-
7,8-
dihydro-pyrido[2,3-d]pyrimidin-6-yl}benzoic acid hydrazide (7, 30 mg, 0.06
mmol) was
suspended in triethyl orthoformate (5 mL) to which was added trifluoroacetic
acid (1 mL).
The resulting reaction mixture was heated at 130 C for 2 h. The volatiles
were removed
and the residue was taken up in dichloromethane (1 x 20 mL) and washed with
10% sodium
hydroxide solution (2 x 10 mL). The organic layer was dried over sodium
sulfate, filtered
and evaporated. The residue was purified by column chromatography using
dichloromethane:methanol (95:5). The title compound (18 mg, 0.03 mmol, 50 %)
was
obtained as a yellow solid. ESMS m/z 543 (M+H)+; 1H NMR (400 MHz, CDC13) 6 ppm
8.57 (s, 1H) 8.50 (s, 1H) 8.21 (d, J= 1.5 Hz, 1H) 8.04 (dd, J= 8.0, 1.5 Hz,
1H) 7.62 (s, 1H)
7.52 - 7.60 (m, 3H) 7.29 (br. s., 1H) 6.98 (d, J= 9.0 Hz, 2H) 4.50 (q, J= 6.8
Hz, 2H) 3.16 -
3.27 (m, 4H) 2.56 - 2.65 (m, 4H) 2.37 (s, 3H) 1.39 (d, J= 6.8 Hz, 3H).
Example 2: Synthesis of 6-[2-chloro-4-(thiophen-2-yl)phenyl]-8-ethyl-2-(4-(4-
methylpiperazin-1-yl)phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (13)
Br Br
\ jl ~ ~ N \ \ ~ N \ \
S N CI CI
S N N 0 \S/`N N 0
0
9 10 11
Br
NI N"') S
N \ I ,,, % \ CI
/ I J\ \ \ \ CI
'
H N N O ~/\NN N O
H
12 13
[00413] Preparation of Intermediate Compounds:
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[00414] Intermediate 2: Synthesis of ethyl 4-bromo-2-chlorophenylacetate
OH OH Br
Br CI Br CI Br CI
14 15 16
~ C N I ~ OH ~ per/
Br CI Br / CI C
17 Br / CI O
18 19
[00415] Step 1: Synthesis of (4-Bromo-2-chlorophenyl)methanol (15)
[00416] 4-Bromo-2-chlorobenzoic acid (14, 92.0 g, 0.39 mol) was dissolved in
dry
tetrahydrofuran (920 mL) and cooled to -15 C. Isobutyryl choroformate (51.0
mL, 0.39
mol) was added followed by N-methylmorpholine (43.5 mL, 0.39 mol). The
resulting
mixture was stirred for 10 minutes at -15 C, cooled to -25 C and the
precipitated N-
methylmorpholine hydrochloride salt was filtered off. The filtrate was warmed
to -5 C and
a solution of sodium borohydride (22.19 g, 0.586 mol) in water (190 mL) was
added
dropwise to the mixture keeping the temperature below 0 C. After stirring for
1 h at 0 C,
the volatiles were evaporated, and the residue was diluted with water (500 mL)
and
dichloromethane (450 mL). The layers were separated and the aqueous layer was
extracted
with dichloromethane (150 mL). The combined organic layers were washed with
water (150
mL), dried over sodium sulfate and evaporated. The product (86.1 g, 0.39 mol,
99%) was
obtained as a white crystalline solid.
[00417] Step 2: Synthesis of 4-Bromo-l-bromomethyl-2-chlorobenzene (16)
[00418] Phosphorus tribromide (40.5 mL, 0.431 mol) was added dropwise to a
solution
of (4-bromo-2-chlorophenyl)-methanol (15, 86.1 g, 0.386 mol) in dichloroethane
(430 mL)
at 0 C. The reaction mixture was stirred for 10 minutes at this temperature
then for 0.5 h at
10 C. The mixture was cooled to 0 C and a sodium hydroxide solution (600 mL,
2N) was
added dropwise. The two layers were separated and the aqueous layer was
extracted with
dichloroethane (200 mL). The combined organic layers were washed with water
(200 mL),
dried over sodium sulfate and evaporated in vacuo. The crude product (91 g)
was distilled
under reduced pressure (7 mmHg), to give 4-bromo-l-bromomethyl-2-chlorobenzene
(62.5
g, 0.22 mol, 57 %) as a colorless oil.
[00419] Step 3: Synthesis of (4-Bromo-2-chlorophenyl)acetonitrile (17)
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[00420] To a stirred solution of 4-bromo-l-bromomethyl-2-chlorobenzene (16,
62.5 g,
0.22 mol) in dichloroethane (522 mL) and water (480 mL) was added
tetrabutylammonium
chloride (5.05 g), followed by a solution of potassium cyanide (43.2 g, 75.8
mmol) in water
(523 mL). and the solution was stirred for 4 h at room temperature. The layers
were
separated and the aqueous layer was extracted with dichloroethane (100 mL).
The combined
organic layers were washed with water (100 mL), dried over sodium sulfate
filtered and
evaporated. The crude product (52 g) was distilled under reduced pressure (1
mmHg),
affording (4-bromo-2-chlorophenyl)-acetonitrile (45.5 g, 0.220 mol, 90 %).
[00421] Step 4: Synthesis of (4-bromo-2-chlorophenyl)acetic acid (18)
[00422] (4-Bromo-2-chlorophenyl)acetonitrile (17, 45.5 g, 0.22 mol) was added
to 675
mL sodium hydroxide solution (8.2 %) and heated at reflux for 4 h. The
homogeneous
solution was cooled to room temperature and concentrated hydrochloric acid
(117 mL) was
added. The mixture was extracted with dichloromethane (500, 200 mL). The
combined
organic layers were washed with water (100 mL), dried over sodium sulfate and
filtered.
The filtrate was treated with charcoal (4.5 g), filtered and evaporated. The
residue was
triturated with hexane (200 mL) and the solid was collected to give (4-bromo-2-
chlorophenyl)-acetic acid (44.6 g, 0.18 mol, 81 %) as a white crystalline
solid. ESMS m/z
497 [2M-H]-.
[00423] Step 5: Synthesis of (4-bromo-2-chlorophenyl)acetic acid ethyl ester
(19)
[00424] (4-Bromo-2-chlorophenyl)-acetic acid (18, 44.03 g, 0.18 mol) was
dissolved in
ethanol (440 mL) and thionyl chloride (44.0 mL, 0.61 mol) was added dropwise.
The
mixture was refluxed for 1 h, and evaporated. The residue was taken up in
toluene and
evaporated (2 x 100 mL). The crude oily product was dissolved in
dichloromethane (300
mL) and washed with water (2 x 100 mL), dried over sodium sulfate, filtered
and
evaporated. The residue was dried in high vacuum (0.2 mmHg) at room
temperature,
solidifying to a light yellow low melting crystalline solid (45.5 g, 0.16 mol,
93 %) ESMS
m/z 294 [M+H+NH3]+;1H NMR (300 MHz, CDC13): 6 7.55 (d, J= 2 Hz, 1H), 7.37 (dd,
J=
8, 2 Hz, 1H), 7.16 (d, J= 8 Hz, 1H), 4.18 (q, J= 7 Hz, 2H), 3.71 (s, 2H), 1.26
(t, J= 7 Hz,
3H).
[00425] Synthesis of 6-[2-chloro-4-(thiophen-2-yl)phenyl]-8-ethyl-2-(4-(4-
methyllpiperazin-1-yl)phenylamino p r~ ido[2,3-d]pyrimidin-7(8H)-one (13)
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Br Br
S N NH
\S N N 0 CI N N 0 CI
0
9 10 11
Br N) / I I S
CI
H N N O NN N 0 CI
H
12 13
[00426] Step 1: Synthesis of 6-(4-bromo-2-chlorophenyl)-8-ethyl-2-
(methylthio)pyrido[2,3-d]pyrimidin-7(8H)-one (10)
[00427] To a solution of 4-ethylamino-2-(methylthio)pyrimidine-5-carbaldehyde
(9, 1.00
g, 5.07 mmol) in anhydrous dimethylacetamide (10 mL) was added ethyl 4-bromo-2-
chlorophenylacetate (1.70 g, 6.12 mmol) and cesium carbonate (3.30 g, 10.13
mmol). The
reaction mixture was stirred at 100 C for 2 h. The mixture was poured into
ice water and
the orange solid was collected, washed with water, dried and purified by
Teledyne-Isco
using a hexane:ethyl acetate gradient (1:0 -* 4:1) to afford the title
compound as a white
solid (0.30 g, 0.73 mmol, 14%). ESMS m/z 410 (M+H)+; 1H NMR (400 MHz, CDC13) 6
ppm 8.65 (s, 1H), 7.66 (d, J= 1.5 Hz, 1H), 7.63 (s, 1H), 7.47 (dd, J= 8.3, 1.5
Hz, 1H), 7.26
(d, J= 8.3 Hz, 1H), 4.55 (q, J= 7.0 Hz, 2H), 2.66 (s, 3H), 1.37 (t, J= 7.0 Hz,
3H).
[00428] Step 2: Synthesis of 6-(4-bromo-2-chlorophenyl)-8-ethyl-2-
(methylsulfinyl)pyrido [2,3-d] pyrimidin-7(8H)-one (11)
[00429] To a solution of 6-(4-bromo-2-chlorophenyl)-8-ethyl-2-(methylthio)-
pyrido[2,3-
d]pyrimidin-7(8H)-one (10, 1.30 g, 3.16 mmol) in dichloromethane (20 mL) was
added
dropwise a solution of 3-chloroperbenzoic acid (77 %, 0.57 g, 2.54 mmol) in
dichloromethane (5 mL) at 0-5 C and the mixture was stirred for 5 h. The
reaction mixture
was washed with saturated sodium bicarbonate solution (2 x 20 mL) and water
(10 mL), and
the organic layer was dried over sodium sulfate, filtered and evaporated. The
crude product
was purified by silica gel column chromatography using dichloromethane:ethyl
acetate
(5:1-*5:2-*2:1-*1:1) to give the title compound as an off-white solid (0.96 g,
2.25 mmol,
71%). ESMS m/z 426 (M+H)+;
[00430] Step 3: Synthesis of 6-(4-bromo-2-chlorophenyl)-8-ethyl-2-(4-(4-
methylpiperazin-1-yl)-phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (12)
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[00431] 6-(4-Bromo-2-chlorophenyl)-8-ethyl-2-(methylsulfinyl)pyrido [2,3 -
d]pyrimidin-
7(8H)-one (11, 0.60 g, 1.41 mmol) and 4-(4-methylpiperazino)aniline (0.27 g,
1.41 mmol)
were stirred at 150 C for 4 h. The cooled reaction mixture was taken up in
dichloromethane
(50 mL) and washed with 10% NaOH (1 x 25 mL) then with water (1 x 20 mL). The
organic layer was dried over sodium sulfate, filtered and evaporated. The
residue was
purified by column chromatography using chloroform: methanol (100:3) as eluent
to give
the title compound as a yellow solid (0.32 g, 0.58 mmol, 41%). ESMS m/z 553
(M+H)+; 1H
NMR (400 MHz, CDC13) 6 ppm 8.53 (s, 1H), 7.65 (d, J= 1.8 Hz, 1H), 7.52 - 7.59
(m, 3H),
7.45 (dd, J= 8.2, 1.9 Hz, 1H), 7.28 (d, J= 8.2 Hz, 1H), 6.97 (d, J= 8.8 Hz,
2H), 4.48 (q, J
= 7.0 Hz, 2H), 3.17 - 3.26 (m, 4H), 2.55 - 2.70 (m, 4H), 2.37 (s, 3H), 1.37
(t, J= 7.0 Hz,
3H).
[00432] Step 4: Synthesis of 6-[2-chloro-4-(thiophen-2-yl)phenyl]-8-ethyl-2-(4-
(4-
methylpiperazin-1-yl)phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (13)
[00433] 6-(4-Bromo-2-chlorophenyl)-8-ethyl-2-(4-(4-methylpiperazin-l-
yl)phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (12, 50 mg, 0.09 mmol),
thiophene-2-
boronic acid (35 mg, 0.27 mmol), K3PO4 (57 mg, 0.27 mmol) and PdC12(dppf) (7
mg, 0.01
mmol) were mixed as solids and placed under argon. Argon was bubbled through a
mixture
of dimethylformamide:water (20:1, 2.0 mL) for 20 min. The solvent was added to
the solid
and the suspension was heated under microwave irradiation at 140 C for 30
min. The
reaction mixture was evaporated and the crude product was purified by column
chromatography, eluting with dichloromethane:methanol (100:3). The product was
triturated with refluxing acetonitrile to yield the title compound as a yellow
solid (48 mg,
0.09 mmol, 100 %). ESMS m/z 557 (M+H)+; 1H NMR (400 MHz, CDC13) 6 ppm 8.54 (s,
1H), 7.72 (s, 1H), 7.51 - 7.60 (m, 4H), 7.40 (d, J= 8.0 Hz, 1H), 7.30 - 7.36
(m, 2H), 7.27 (s,
1H),7.08-7.13(m, 1H),6.97(d,J=8.8Hz,2H),4.50(q,J=7.0Hz,2H),3.16-3.28(m,
4H), 2.54 - 2.69 (m, 4H), 1.39 (t, J= 7.0 Hz, 3H).
[00434] Step 4': Synthesis of 6-[2-chloro-4-(thiophen-2-yl)phenyl]-8-ethyl-2-
(4-(4-
methylpiperazin-1-yl)phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-one
hydrochloride
(13)
[00435] 6-(4-Bromo-2-chlorophenyl)-8-ethyl-2-(4-(4-methylpiperazin-l-
yl)phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (12, 666 mg, 1.2 mmol),
thiophene-2-
boronic acid (192 mg, 1.5 mmol), NaHCO3 (504 mg, 6 mmol), Pd(PPh3)4 (50 mg),
dioxane
(30 mL) and water (6 mL) were placed in a microwave tube and oxygen was
removed by a
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stream of argon. The reaction was heated by microwave at 140 C for 1.5 h and
was
monitored by LC/MS. The reaction mixture was evaporated and the solid was
extracted
with chloroform (100 mL). The solids were removed and the filtrate was
evaporated and
purified by silica gel chromatography (CHC13 + 5% MeOH). Yield 82 mg, 5% as a
yellow
solid. LCMS m/z 557 (M+H)+, Rt 1.84min; 1H NMR (400 MHz, CDC13) 6 ppm 8.56 (s,
1H), 7.73 (d, 1H), 7.54 - 7.60 (m, 4H), 7.33 - 7.42 (m, 4H), 7.10 - 7.12 (q,
1H), 6.98-7.00
(d, 2H), 4.49-4.54 (q, 2H), 3.22 - 3.25 (m, 4H), 2.61 - 2.63 (m, 4H), 2.38 (s,
3H), 1.38 - 1.42
(t, 3H).
[00436] The hydrochloride salt was prepared by addition of an excess of
hydrogen
chloride in dioxane to a solution of free base in chloroform with quantitative
yield. LCMS
m/z 557 (M+H)+; Rt 1.84min; 1H NMR (400 MHz, DMSO-d6) 6 ppm 10.95 - 11.15 (s,
1H),
10.05 - 10.15 (s, 1H), 8.80 (s, 1H), 7.88 (s, 1H), 7.82 (s, 1H), 7.71 - 7.73
(d, 2H), 7.65 - 7.67
(m, 2H), 7.62 - 7.64 (d, 1H), 7.43 - 7.45 (d, 1H), 7.17-7.19 (m, 1H), 7.03 -
7.05 (d, 2H),
4.33-4.38 (q, 2H), 3.75 - 3.78 (d, 2H), 3.47 - 3.49 (d, 2H), 3.07 - 3.21 (m,
4H), 2.80 (s, 3H),
1.26 - 1.29 (t, 3H).
[00437] Examples 3-5:
[00438] The following compounds were made by the method of Example 2 using the
appropriate arylacetic acid at Step 1 and aniline at Step 3. Examples
containing secondary
amines on the aniline were synthesized using the appropriate Boc protected
aminoaniline
and in the final step were treated with a solution of hydrogen chloride in an
organic solvent
to produce the example compound, usually isolated as the hydrochloride salt.
In this
manner, Example 3 was prepared using methyl 2-[5-methyl-2-(n-tert-
butoxycarbonylpiperidine)- 1, 3 -thiazol-4-yl] acetate and 4-(4-
methylpiperazino)aniline.
Example 4 and Example 5 were prepared from Example 3 by reductive methylation
and
treatment with acetic anyhydride respectively.
Ex. Structure MW LCMS LCMS Rt
Method Ion
NH
N-
3 ~~ s 544.7 A 545 0.96
v \~~
H
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N
4 \\ \ S 558.8 A 560 0.98
H
O\\y-
N
N - S 586.8 A 587 1.16
A~kr_ ~__O_
H
[00439] Examples 6-33:
[00440] Preparation of Intermediate Compounds:
[00441] Intermediate 3: Synthesis of 2-(4-Amino-phenyl)-morpholine-4-
carboxylic
5 acid tert-butyl ester
O OH
H
Br I \ N\~\OH
-OW / -OWN I /
O O O
1 2 3
O J<
OH
N NyO N_f,-O
---\OH O
N+
O\ N
/[\
O
O
4 5 6
[00442] Step 1: Synthesis of 2-(4-nitro-phenyl)-oxirane (2).
[00443] To an ice-cold stirred suspension of 4-nitrophenacyl bromide (80 g,
0.33 mol) in
methanol (800 mL) was added sodium borohydride (13.64 g, 0.36 mol) in small
portions.
After stirring for 2 h at 0-5 C, potassium carbonate (45.20 g, 0.33 mol) was
added in small
portions at the same temperature. The suspension was stirred for 18 h at room
temperature,
diluted with brine (600 mL) and extracted with diethyl ether (600 mL, 500 mL),
the
combined organic layers were dried over sodium sulfate, filtered and
evaporated. The title
compound (54.87 g, 0.33 mmol, 100%) was obtained as a pale yellow solid.
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[00444] Step 2: Synthesis of 2-(2-hydroxy-ethylamino)-1-(4-nitro-phenyl)-
ethanol
(3).
[00445] The mixture of 2-(4-nitro-phenyl)-oxirane (24.1 g, 0.15 mol) in
ethanolamine
(500 mL) was stirred at 40 C for 2 h, then room temperature for 18 h. The
reaction was
partitioned between ethyl acetate (200 mL) and water (200 mL) and the aqueous
layer was
extracted with ethyl acetate (4 x 100 mL). The combined organic layers were
dried over
sodium sulfate, filtered and evaporated. The residue was triturated with
acetonitrile and
collected to give the title compound (19.80 g, 0.09 mmol, 60%) as a white
solid. ESMS m/z
227 (M+H)+;
[00446] Step 3: Synthesis of (2-hydroxyethyl)-[2-hydroxy-2-(4-nitro-phenyl)-
ethyl]-
carbamic acid teat-butyl ester (4).
[00447] To 2-(2-hydroxy-ethylamino)-1-(4-nitro-phenyl)-ethanol (10.00 g, 44.2
mmol) in
dichloromethane (80 mL) was added triethylamine (6.15 mL, 4.46 g, 44.2 mmol)
followed
by di-tert-butyl dicarbonate (9.65 g, 44.2 mmol) dissolved in dichloromethane
(20 mL). The
reaction mixture was stirred for 4 h at room temperature, washed with water
(50 mL), and
the aqueous layer was back extracted with ethyl acetate (2 x 50 mL). The
combined organic
layers were dried over sodium sulfate, filtered and evaporated. The residue
was triturated
with diisopropyl ether and collected. The title compound (12.04 g, 36.9 mmol)
was obtained
as a white solid. ESMS m/z 349 (M+Na)+;
[00448] Step 4: Synthesis of 2-(4-nitro-phenyl)-morpholine-4-carboxylic acid
tert-
butyl ester (5).
[00449] To an ice-cold stirred mixture of (2-hydroxyethyl)-[2-hydroxy-2-(4-
nitro-
phenyl)-ethyl]-carbamic acid tert-butyl ester (5.50 g, 16.8 mmol) and
triphenylphosphine
(5.17 g, (19.7 mmol) in toluene (80 mL) was added triethylamine (6.15 mL, 4.46
g, 44.2
mmol) followed a solution of of di-tert-butylazodicarboxylate (3.10 mL, 19.7
mmol) in
toluene (30 mL) dropwise. The reaction mixture was stirred for 18 h at room
temperature,
washed with water (50 mL), and the aqueous layer was back extracted with ethyl
acetate (2
x 50 mL). The combined organic layers were dried over sodium sulfate, filtered
and
evaporated. The residue was purified by column chromatography eluting with
dichloromethane, the obtained product was triturated with diisopropyl ether
and collected.
The title compound (3.56 g, 11.5 mmol, 68%) was obtained as a white solid.
ESMS m/z 253
(M+H-tBu)+; 1H NMR (400 MHz, CDC13) 6 ppm 8.23 (d, J= 8.3 Hz, 2H) 7.57 (d, J=
8.3
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Hz, 2H) 4.53 (d, J= 10.3 Hz, 1H) 4.20 (br. s., 1H) 4.06 (d, J= 11.3 Hz, 1H)
3.94 (br. s.,
1H) 3.66 - 3.75 (m, 1H) 3.06 (br. s., 1H) 2.76 (br. s., 1H) 1.50 (s, 9H).
[00450] Step 5: Synthesis of 2-(4-amino-phenyl)-morpholine-4-carboxylic acid
tert-
butyl ester (6).
[00451] A mixture of 2-(4-nitro-phenyl)-morpholine-4-carboxylic acid tert-
butyl ester
(20 mg, 0.064 mmol) and Pd/C (5%, 2 mg) in methanol was stirred under hydrogen
for 24
h. The catalyst was filtered off and washed with methanol. The filtrate was
evaporated to
give the title compound as an off-white solid (14 mg, 0.050 mmol, 78%). ESMS
m/z 223
(M+H-tBu)+; 1H NMR (400 MHz, DMSO-d6) 6 ppm 7.00 (d, J= 8.3 Hz, 2H) 6.52 (d,
J=
8.3 Hz, 2H) 5.04 (s, 2H) 4.15 (dd, J= 10.5, 2.5 Hz, 1H) 3.88 (dd, J= 11.8, 2.5
Hz, 1H) 3.76
(d, J= 12.8 Hz, 2H) 3.48 (td, J= 11.7, 2.8 Hz, 1H) 2.92 (br. s., 1H) 2.77 (br.
s., 1H) 1.41 (s,
9H).
[00452] Intermediate 4: Synthesis of 2-(4-Amino-phenyl)-thiomorpholine-4-
carboxylic acid tert-butyl ester
~r l
11 S
\O S O N
W N+
O O N , 11
O
1 2 3
S OO S~
N JD N `r N `(~
O
\ H2N H2N
4 5 6
[00453] Step 1: Synthesis of [2-nitro-l-(4-nitrophenyl)-ethylsulfanyl] -acetic
acid
methyl ester (2).
[00454] To an ice-cold stirred solution of 1-nitro-4-(2-nitro-vinyl)-benzene
(5.26 g, 27.09
mmol) and triethylamine (4.45 ml, 31 mmol) in tetrahydrofuran (80 mL)
mercaptoacetic
acid methyl ester (2.75 mL, 30.7 mmol) was added in one portion. The reaction
mixture was
stirred for 3 min, and cc. HC1(3.55 mL) was added. The precipitated
triethylamine HCl salt
was removed by filtration through Perlite. The filtrate was evaporated and the
residue was
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taken up in dichloromethane (100 mL), washed with 1M HCl (20 mL), water (2 x
20 mL),
dried over sodium sulfate and evaporated. The title compound was obtained as a
pink low
melting point crystalline material (8.11 g, 27 mmol, 99%). ESMS m/z 323
(M+Na)+.
[00455] Step 2: Synthesis of 6-(4-aminophenyl)-thiomorpholin-3-one (3)
[00456] Acetic acid (240 mL) was heated to 72 C, then Zn (25.38 g, 388 mmol)
and [2-
nitro-1-(4-nitrophenyl)-ethylsulfanyl] -acetic acid methyl ester (3.00 g,
10.00 mmol) were
added in one portion. The well-stirred suspension was heated at reflux for 0.5
h, filtered
through activated charcoal and evaporated. Dichloromethane (50 mL), water (30
mL) and
10% aqueous NaOH (50 mL) were added. The suspension was filtered through
Perlite, the
layers were separated and the aqueous layer was extracted with dichloromethane
(2 x 20
mL). the combined organic layers were washed with water (10 mL), dried over
sodium
sulfate and evaporated. The residue was triturated with chloroform (10 mL) and
the solid
was collected and washed with chloroform (2 mL) to obtain the title product
(0.225 g, 1.08
mmol, 11 %). ESMS m/z 209 (M+H)+.
[00457] Step 3: Synthesis of 6-(4-aminophenyl)-thiomorpholine (4)
[00458] To the solution of 6-(4-aminophenyl)-thiomorpholin-3-one (0.26 g, 1.25
mmol)
in anhydrous tetrahydrofuran (6.7 mL) was added lithium aluminium hydride
(0.16 g, 4.27
mmol) in several portions and the mixture was stirred at 60 C for 2 h. Na2SO4
x 10 H2O
(2.00 g) was added in small portions until the complex was decomposed. The
suspension
was filtered, the solids were washed with tetrahydrofuran (2 x 3 mL) and the
combined
filtrates were evaporated. The title compound was obtained as a viscous oil
(0.25 g, 1.26
mmol, 100 %). ESMS m/z 195 (M+H)+.
[00459] Step 4: Synthesis of 2-(4-aminophenyl)-thiomorpholine-4-carboxylic
acid
teat-butyl ester (5)
[00460] To 6-(4-aminophenyl)-thiomorpholine (0.25 g, 1.26 mmol) in anhydrous
tetrahydrofuran (2.5 mL) was added di-tert-butyl dicarbonate (0.25 g, 1.14
mmol). The
solution was stirred at room temperature for 1 h. After evaporation, the
residue was purified
by column chromatography using hexane:ethyl acetate (4:1 -* 3:1 -* 2:1). The
title
compound was obtained as a white crystalline solid (0.13 g, 0.42 mmol, 33 %).
ESMS m/z
317 (M+Na)+;. 1H NMR (400 MHz, DMSO-d6) 6 ppm 6.99 (d, J= 8.3 Hz, 2H) 6.51 (d,
J=
8.3 Hz, 2H) 5.07 (s, 2H) 4.23 (d, J= 13.6 Hz, 1H) 4.12 (br. s., 1H) 3.69 (dd,
J= 10.8, 2.8
Hz, 1H) 3.13 (br. s., 1H) 2.98 (br. s., 1H) 2.73 (td, J= 12.7, 3.0 Hz, 1H)
2.56 (d, J= 13.6
Hz, 1H) 1.40 (s, 9H).
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[00461] Step 5: Synthesis of 2-(4-aminophenyl)-thiomorpholine-S,S-dioxide-4-
carboxylic acid tent-butyl ester (6).
To a solution of 2-(4-amino-phenyl)-thiomorpholine-4-carboxylic acid tert-
butyl ester (150
mg, 0.51 mmol) in dichloromethane (10 mL) was added a solution of 3-
chloroperbenzoic
acid (77 %, 258 g, 1.15 mmol) in dichloromethane (11 mL) at 0-5 C and the
mixture was
stirred at 0-5 C for 1.5 h. The reaction mixture was washed with 10% aqueous
sodium
bicarbonate (20 mL), the organic layer was dried over sodium sulfate, filtered
and
evaporated. The residue was purified by column chromatography using
dichloromethane:methanol (100:2). The title compound was obtained as a pale
yellow solid
(120 mg, 0.37 mmol, 72%). ESMS m/z 271 (M+H-tBu)+.
[00462] Intermediates 6-10:
[00463] The following compounds were made by the method of Example 2 using the
appropriate arylacetic acid at Step land aniline at Step 3. Examples
containing secondary
amines on the aniline were synthesized using the appropriate Boc protected
aminoaniline
and in the final step a small portion was treated with a solution of hydrogen
chloride in an
organic solvent to produce the example compound, usually isolated as the
hydrochloride
salt.
Int. Structure MW LCMS LCMS Rt
Method Ion
5 I , 519.5 A 519 1.39
O
H
H C\ 6 a 540.9 A 540 1.38
N, O
H
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m
s
7 a 556.9 A 556 1.49
N, C~O
H
~S /
H \ \ \ \
8 o ,C, a 570.9 A 572 1.51
W O q
H \ \ \ \
9 I / 0 570.9 A 570 1.68
O
H
[00464] Intermediate 10: Synthesis of 6-(4-bromo-2-chlorophenyl)-8-ethyl-2-[4-
(4-
methyl-thiomorpholin-2yl)-phenylamino]-8H-pyrido [2,3-d] pyrimidin-7-one
hydrochloride (2)
/ Br Br
HN N INII \ \ \
2HC1 \ I / CI \ I / CI
N N N O N N N O
H H
xHCI
7 10
[00465] To a suspension of 6-(4-bromo-2-chlorophenyl)-8-ethyl-2-(4-
thiomorpholin-2-
yl-phenylamino)-8H-pyrido[2,3-d]pyrimidin-7-one dihydrochloride (40 mg, 0.06
mmol) in
dichloromethane (1 mL) was added triethylamine (18 L, 12.9 mg, 0.13 mmol),
benzotriazole (8 mg, 0.07 mmol) and formaldehyde (37%, 5.7 L, 0.08 mmol) and
the
reaction was stirred at room temperature for 2 h. Sodium borohydride (4.5 mg,
0.12 mmol)
was added and the reaction mixture was stirred for 18 h, diluted with
dichloromethane (10
mL) and washed with sodium bicarbonate solution (10%, 10 mL). The aqueous
layer was
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back extracted with dichloromethane (5 x 5 mL), and the combined organic
layers were
washed with water (10 mL), dried over sodium sulfate, filtered and evaporated
in vacuo.
The crude product was purified by column chromatography eluting with
dichloromethane:2-
propanol (100:5) to give a white solid (6.8 mg, 0.01 mmol, 17 %). The free
base was
dissolved in dichloromethane (3 mL), treated with HC1/diethyl ether (0.445 M,
27 l, 0.01
mmol), stirred at room temperature for 18 h and evaporated to give the title
compound as a
solid (7.7 mg, 0.01 mmol, 100%). ESMS m/z 570/572 (M+H)+; 1H NMR (400 MHz,
DMSO-d6) 6 ppm 10.00 (s, 1H) 8.82 (s, 1H) 7.82 - 7.89 (m, 3H) 7.78 (s, 1H)
7.61 (dd, J=
8.4, 1.6Hz, 1H) 7.33 - 7.41 (m, 3H) 4.30 - 4.48 (m, 3H) 3.60 (br. s., 2H) 3.24
(br. s., 2H)
2.93 (br. s., 2H) 2.77 (br. s., 3H) 1.32 (t, J= 6.9Hz, 3H).
[00466] Intermediate 11:
[00467] The following compound was made by the method of Intermediate 10
starting
with intermediate 6.
Int. Structure MW LCMS LCMS Rt
Method Ion
g
o
11 ~I G 554.9 A 556 1.42
rv' ~ o
H
[00468] Examples 6-33:
[00469] The following compounds were made by the method of Example 2 using the
appropriate arylacetic acid at Step 1, aniline at Step 3 and boronic acid or
ester at Step 4.
Examples containing secondary amines on the aniline were synthesized using the
appropriate Boc protected aminoaniline and in the final step were treated with
a solution of
hydrogen chloride in an organic solvent to produce the example compound,
usually isolated
as the hydrochloride salt. Reaction of 12 with cyclopropylboronic acid
generated a mixture
of Examples 7 and 8 which were separated.
Ex. Structure MW LCMS LCMS Rt
Method Ion
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6 480.6 A 481 1.41
H
515.1 A 515 1.45
7 II I AG
1~ Cal
H
8 520.7 A 521 1.49
H
S
9 \\ \ \ 557.1 A 557 1.47
\%\N L a
0
H
\\ 522.7 A 523 1.47
v
H
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\N~ /
N
11 \ =-N 571.1 A 571 1.60
~,a
H
I \
\N~ / I O
\
12 \ cx \ O 541.1 A 541 1.45
/ ci
W, I H
O
N
13 \ 541.1 A 541 1.44
0
Cl
H
/ N
14 \\ \ \ \ 552.1 A 552 1.06
H
\ ^ / \ N
N' 11
15 \ \ \ \ 552.1 A 552 1.16
H
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\~ / \ IIN
16 \ I\ \ \ 553.1 A 553 1.21
N^ / o ci
H
N
N
17 I \ \ \ 540.0 A 540 1.20
HN
H
N
~ / \ IIN
18 /N \ \ \ \ 554.1 A 554 1.24
H
N
NH
19 / \ \ \ 506.6 B 507 1.38
H J
N
\~ / I H
20 506.6 B 507 1.39
N O
H J
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N
N
21 555.1 B 555 1.58
ci
0
H
/
N
N
22 520.6 B 521 1.58
H
\N C
NH
23 \ \ \ \ 590.1 B 590 1.84
H
\N l \ \\ N
OI
24 N \ / 602.1 B 602 1.54
N~N O
H
\N
25 N \ II / \ 520.6 B 521 1.52
H
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s
IN \
26 / \ \ 522.7 B 523 1.85
v \~/ o
H
CI N
27 570.1 B 570 1.71
O
H
O
28 506.6 B 507 1.76
H
O\
\~ / \ N
29 // \ \ \ 547.7 B 548 1.65
H
H
\~ / \ N O
30 / N~ \ \ 609.1 B 609 1.47
CI
H I
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N
31 518.6 B 519 1.48
H J
~,N N
32 " \ "~ \ \ 567.7 B 568 1.53
0
H
17", 0-1
\ ^ / \ N
33 / \ \ \ 582.1 B 582 1.69
wj~
0
H
[00470] Example 34: Synthesis of 6-[2-chloro-4-(5-methylthiazol-2-yl)phenyl]-8-
ethyl-2-(4-(4-methylpiperazin-1-yl)phenylamino)pyrido [2,3-d] pyrimidin-7(8H)-
one
(20)
N/~ Br
IIII S
1 I N \ I \N
\ CI N / I N \ \ \
H N N O a CI
H N N~
12 20
[00471] 6-(4-Bromo-2-chlorophenyl)-8-ethyl-2-(4-(4-methylpiperazin-l-
yl)phenylamino)pyrido[2,3-d]pyrimidin-7(8H)-one (12, 194 mg, 0.35 mmol), 5-
methyl-2-
(tributylstannyl)-thiazole (171 mg, 0.44 mmol), Pd(PPh3)4 (50 mg) and toluene
(15 mL)
were placed in a microwave tube and oxygen was removed by a stream of argon.
The
reaction was heated by microwave at 140 C for 1.5 h and was monitored by
LGMS. The
reaction mixture was evaporated and the solid was extracted with chloroform
(100 mL). The
solids were removed and the filtrate was evaporated and purified by silica gel
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chromatography (CHC13 + 5% MeOH). Yield 17mg, 0.03 mmol, 8% as yellow solid.
LCMS
m/z 572 (M+H)+Rt 1.74min; 1H NMR (400 MHz, CDC13) 6 ppm 8.56 (s, 1H), 8.04 (s,
1H),
7.81 - 7.83 (d, 1H), 7.54 - 7.61 (m, 4H), 7.46 - 7.48 (d, 1H), 7.32 - 7.37 (s,
1H), 6.97-7.00
(d, 2H), 4.48-4.54 (q, 2H), 3.24 - 3.26 (m, 4H), 2.63 - 2.65 (m, 4H), 2.54 (s,
3H), 2.40 (s,
3H), 1.38 - 1.42 (t, 3H).
[00472] Examples 35-37:
[00473] The following compounds were made by the method of Example 34 using
the
appropriate heteroarylstannane. Examples containing secondary amines on the
aniline were
synthesized using the appropriate Boc protected aminoaniline and in the final
step were
treated with a solution of hydrogen chloride in an organic solvent to produce
the example
compound, usually isolated as the hydrochloride salt.
Ex. Structure MW LCMS LCMS Rt
Method Ion
N
35 558.1 B 558 1.65
H J
s-
N
36 523.7 B 524 1.57
~~ / I fV
37 558.1 B 558 1.60
H
[00474] Example 38: Synthesis of 6-[2,2']bithiophenyl-5-yl-8-ethyl-2-[4-(4-
methyl-
piperazin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidin-7-one. (22)
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N N~1 QH
N \ I Br N ao,,, B-OH
H N N O H N N O
21
CN,, ao,;, N
N N 0
H
22
[00475] Step 1: Synthesis of 8-ethyl-2-[4-(4-methyl-piperazin-1-yl)-
phenylamino]-7-
oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-boronic acid. (21)
6-Bromo-8-ethyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-8H-pyrido[2,3-
d]pyrimidin-
5 7-one (5, 100 mg, 0.22 mmol), bis(pinacolato)diboron (63 mg, 0.25 mmol),
potassium
acetate (66 mg, 0.68 mmol) and PdC12(PPh3)2 (16 mg, 0.02 mmol) were mixed
under argon
in degassed toluene (3 mL). The resulting suspension was irradiated for 30 min
at 120 C in
a microwave reactor. After completion, the reaction mixture was evaporated and
the residue
was purified by column chromatography using dichloromethane: methanol:
triethylamine
(4:1:0-*1:1:0-*0:95:5) as eluent. The crude product was dissolved in
dichloromethane (10
mL), washed with water (5 mL), and the organic layer was dried over sodium
sulfate,
filtered and evaporated. The title compound (15 mg, 0.04 mmol, 18 %) was
obtained as a
yellow solid. ESMS m/z 409 (M+H)+; 1H NMR (400 MHz, DMSO-d6) 6 ppm 10.07 (br.
s.,
1H) 8.85 (s, 1H) 8.52 (s, 2H) 8.28 (s, 1H) 7.64 (br. s., 2H) 6.94 (d, J= 9.0
Hz, 2H) 4.33 (q,
J = 6.9 Hz, 2H) 3.07 - 3.14 (m, 4H) 2.43 - 2.47 (m, 4H) 2.22 (s, 3H) 1.26 (t,
J = 6.9 Hz,
3H).
[00476] Step 2: Synthesis of 6-[2,2']bithiophenyl-5-yl-8-ethyl-2-[4-(4-methyl-
piperazin-1-yl)-phenylamino]-8H-pyrido[2,3-d]pyrimidin-7-one. (22)
8-Ethyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-
pyrido[2,3-d]
pyrimidine-6-boronic acid (21, 71 mg, 0.17 mmol), 5-bromo-2,2'-bithiophene (47
mg, 0.19
mmol), sodium carbonate (55 mg, 0.52 mmol) and Pd(PPh3)4 (20 mg, 0.02 mmol)
were
mixed under argon in a degassed mixture of dimethoxyethane:water (10:1, 3 mL).
The
resulting suspension was irradiated for 60 min at 120 C in a microwave
reactor. After
completion, the reaction mixture was evaporated and the residue was purified
by column
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chromatography using dichloromethane:methanol (100:5) as eluent. The crude
product was
triturated with ethyl acetate (10 mL) and collected by filtration. The title
compound (30 mg,
0.057 mmol, 33 %) was obtained as a yellow solid. ESMS m/z 529 (M+H)+; 1H NMR
(400
MHz, DMSO-d6) 6 ppm 9.99 (br. s., 1H) 8.81 (s, 1H) 8.45 (s, 1H) 7.72 (d, J=
3.8 Hz, 1H)
7.66 (br. s., 2H) 7.51 (dd, J= 5.1, 0.9 Hz, 1H) 7.31 - 7.37 (m, 2H) 7.11 (dd,
J= 5.0, 3.8 Hz,
1H)6.95(d,J=9.0Hz,2H)4.42(q,J=7.0Hz,2H)3.04-3.17(m, 4H) 2.42 - 2.48 (m,
4H) 2.23 (s, 3H) 1.31 (t, J= 7.0 Hz, 3H).
[00477] Examples 39-46:
[00478] The following compounds were made by the method of Example 38 using
the
appropriate heteroaryl bromide at Step 2. Examples containing secondary amines
on the
aniline were synthesized using the appropriate Boc protected aminoaniline and
in the final
step were treated with a solution of hydrogen chloride in an organic solvent
to produce the
example compound, usually isolated as the hydrochloride salt.
Ex. Structure MW LCMS LCMS Rt
Method Ion
N~a
39 486.6 A 487 1.46
o
H
I ~~Na S
40 ~I 543.7 A 544 1.39
W- C o
H
\"~ ,"moo
41 528.6 A 529 1.34
N' \ o
H
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42 523.7 A 524 1.29
W-C, 0
H
I ~~Na
43 JI " 523.7 A 524 1.21
\
N' ~ o
H
~N I I
JI \ H-"
44 514.6 A 515 1.13
N' o
H
I
N, N
45 523.7 A 524 1.05
N' \ o
H
~N \
46 JI \ "-0 513.6 A 514 1.33
o
H
Biological Examples
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Example 47 Treatment of Schizophrenia by Administration of a PAK Inhibitor
Compound Disclosed Herein in an Animal Model
[00479] The ability of a PAK inhibitor to ameliorate behavioral and anatomical
symptoms of schizophrenia (i.e., their mouse analogs) is tested in a dominant-
negative
DISCI mouse model of schizophrenia (Hikida et al (2007), Proc Natl Acad Sci
USA,
104(36):14501-14506).
[00480] Forty DISC1 mice (ages 5-8 months) on a C57BL6 strain background are
divided into treatment group (1 mg/kg of compound disclosed herein, oral
gavage) and a
placebo group (0.1% DMSO in physiological saline solution) and analyzed for
behavioral
differences in open field, prepulse inhibition, and hidden food behavioral
tests, with an
interval of about one week between each type of test. In the open field test,
each mouse is
placed in a novel open field box (40 cm X 40 cm; San Diego Instruments, San
Diego, CA)
for two hours. Horizontal and vertical locomotor activities in the periphery
as well as the
center area are automatically recorded by an infrared activity monitor (San
Diego
Instruments). Single breaks are reported as "counts." In this behavioral test,
a significant
reduction in total activity in the treatment group relative to the placebo
group indicates a
possible treatment effect.
[00481] In the hidden food test, mice are food-deprived for 24 h. After
habituation to a
new cage for 5 min, a food pellet is hidden under the cage bedding. The time
it takes for the
mouse to find the food pellet is measured until a maximum of 10 min is
reached. In this
behavioral test, a significant reduction in time to find the food pellet in
the treatment group
relative to the placebo group is indicative of a successful treatment effect.
[00482] In the prepulse inhibition test, acoustic startle and prepulse
inhibition responses
are measured in a startle chamber (San Diego Instruments). Each mouse is
individuated to
six sets of seven trail types distributed pseudorandomly: pulse-alone trials,
prepulse-pulse
trials, and no-stimulus trials. The pulse used is 120dB and the prepulse is 74
dB. A
significant increase in the prepulse inhibition response in the treatment
group relative to the
placebo group is indicative of a successful treatment effect.
[00483] In the forced swim test, each mouse is put in a large plastic
cylinder, which is
half-filled with room temperature water. The test duration is 6 min, during
which the
swim/immobility times are recorded. In this behavioral test, a significant
reduction in
immobility in the treatment group relative to the placebo group is indicative
of a successful
treatment effect.
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[00484] In order to evaluate the ability of the compounds disclosed herein to
alter brain
morphology, an MRI study is conducted on placebo-treated and treated groups of
DISC 1-
DN mice. In vivo MRI experiments are performed on an 11.7T Bruker Biospec
small
animal imaging system. A three-dimensional, fast-spin echo, diffusion weighted
(DW)
imaging sequence with twin navigation echoes is used to assess the ratio of
lateral ventricle
volume to total brain volume. A decrease in this ratio in the treated group
relative to the
ratio observed in the placebo-group is indicative of a successful treatment
effect.
[00485] Statistical Analysis. Statistical analysis is performed by ANOVA or
repeated
ANOVA. Differences between groups are considered significant at p < 0.05.
Example 48 In Vivo Monitoring of Dendritic Spine Plasticity in Double
Transgenic
GFP-M/DN-DISCI Mice Treated with a PAK Inhibitor Compound Disclosed Herein
[00486] In the following experiment, dendritic spine plasticity is directly
monitored in
vivo by two photon laser scanning microscopy (TPLSM) in double transgenic GFP-
M/DN-
DISC1 mice treated with a compound disclosed herein or a placebo. Mice
(C57BL/6)
expressing GFP in a subset of cortical layer 5 neurons (transgenic line GFP-M
described in
Feng et al, 2000, Neuron 28:41-51) are crossed with DN-DISCI C57BL/6 DN-DISCI
mice
(Hikida et al (2007), Proc Natl Acad Sci USA, 104(36):14501-14506) to obtain
heterozygous transgenic mice, which are then crossed to obtain homozygous
double
transgenic GFPM/DN-DISCI mice used in this study.
[00487] GFP-M/DN-DISCI animals aged 28-61 d are anesthetized using avertin (16
Ug
body weight; Sigma, St. Louis, MO). The skull is exposed, scrubbed, and
cleaned with
ethanol. Primary visual, somatosensory, auditory, and motor cortices are
identified based on
stereotaxic coordinates, and their location is confirmed with tracer
injections (see below).
[00488] Long-term imaging experiments are started at P40. The skull is thinned
over the
imaging area as described in Grutzendler et al, (2002), Nature, 420:812-816. A
small metal
bar is affixed to the skull. The metal bar is then screwed into a plate that
connected directly
to the microscope stage for stability during imaging. The metal bar also
allows for
maintaining head angle and position during different imaging sessions. At the
end of the
imaging session, animals are sutured and returned to their cage. Thirty
animals previously
imaged at P40 are then divided into a control group receiving a 1% sugar
solution (oral
gavage once per day) and a treatment group administered a compound disclosed
herein, in
0.1% DMSO (oral gavage. 1 mg/kg, once per day). During the subsequent imaging
sessions
(at P45, P50, P55, or P70), animals are reanesthetized and the skull is
rethinned. The same
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imaging area is identified based on the blood vessel pattern and gross
dendritic pattern,
which generally remains stable over this time period.
[00489] At the end of the last imaging session, injections of cholera toxin
subunit B
coupled to Alexa Fluor 594 are made adjacent to imaged areas to facilitate
identification of
imaged cells and cortical areas after fixation. Mice are transcardially
perfused and fixed
with paraformaldehyde, and coronal sections are cut to verify the location of
imaged cells.
Sections are then mounted in buffer, coverslipped, and sealed. Images are
collected using a
Fluoview confocal microscope (Olympus Optical, Melville, NY).
[00490] For in vivo two photon imaging, a two-photon laser scanning microscope
is used
as described in Majewska et al, (2000), Pflugers Arch, 441:398-408. The
microscope
consists of a modified Fluoview confocal scan head (Olympus Optical) and a
titanium/sulphur laser providing 100 fs pulses at 80 MHz at a wavelength of
920 nm
(Tsunami; Spectra-Physics, Menlo Park, CA) pumped by a 10 W solid-state source
(Millenia; Spectra-Physics). Fluorescence is detected using photomultiplier
tubes (HC125-
02; Hamamatsu, Shizouka, Japan) in whole-field detection mode. The craniotomy
over the
visual cortex is initially identified under whole-field fluorescence
illumination, and areas
with superficial dendrites are identified using a 20x, 0.95 numerical aperture
lens (IR2;
Olympus Optical). Spiny dendrites are further identified under digital zoom (7-
10x) using
two-photon imaging, and spines 50-200 m below the pial surface are studied.
Image
acquisition is accomplished using Fluoview software. For motility
measurements, Z stacks
taken 0.5-1 m apart are acquired every 5 min for 2 h. For synapse turnover
experiments, Z
stacks of dendrites and axons are acquired at P40 and then again at P50 or
P70. Dendrites
and axons located in layers 1-3 are studied. Although both layer 5 and layer 6
neurons are
labeled in the mice used in this study, only layer 5 neurons send a clear
apical dendrite close
to the pial surface thus, the data will come from spines on the apical tuft of
layer 5 neurons
and axons in superficial cortical layers.
[00491] Images are exported to Matlab (MathWorks, Natick, MA) in which they
are
processed using custom-written algorithms for image enhancement and alignment
of the
time series. For motility measurements (see Majewska et al, (2003), Proc Natl
Acad Sci
USA, 100:16024-16029) spines are analyzed on two-dimensional projections
containing
between 5 and 30 individual images; therefore, movements in the z dimension
are not
analyzed. Spine motility is defined as the average change in length per unit
time
(micrometers per minute). Lengths are measured from the base of the protrusion
to its tip.
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The position of spines are compared on different imaging days. Spines that are
farther than
0.5 m laterally from their previous location are considered to be different
spines. Values
for stable spines are defined as the percentage of the original spine
population present on the
second day of imaging. Only areas that show high signal-to-noise ratio in all
imaging
sessions will be considered for analysis. Analysis is performed blind with
respect to animal
age and sensory cortical area. Spine motility (e.g., spine turnover),
morphology, and density
are then compared between control and treatment groups. It is expected that
treatment with
a compound disclosed herein will rescue defective spine morphology relative to
that
observed in untreated control animals.
Example 49 Treatment of Clinical Depression by Administration of a PAK
Inhibitor
Compound Disclosed Herein in an Animal Model
[00492] A rat olfactory bulbectomy (OBX) model of clinical depression (see,
e.g., van
Riezen et al (1990), Pharmacol Ther, 47(1):21-34; and Jarosik et al (2007),
Exp Neurol,
204(1):20-28) is used to evaluate treatment of clinical depression with a
compound
disclosed herein. Dendritic spine density and morphology are compared in
treated and
untreated groups of animals as described below. It is expected that treatment
of OBX
animals with a PAK inhibitor will cause an increase in spine density relative
to that
observed in untreated OBX animals.
[00493] All experiments are performed in strict accordance with NIH standards
for
laboratory animal use. The study uses 48 adult male Sprague-Dawley rats (230-
280 g)
housed in groups of four animals (two sham and two OBX), as indicated in van
Riezen et al
supra, in a controlled environment with food and water available ad libitum.
Half of the
experimental animals (n = 24) undergo bilateral olfactory bulbectomy (OBX)
while the
other half undergo sham surgery (n = 24). Upon completion of surgery, animals
are allowed
to recover for 2 weeks prior to behavioral testing. This is necessary to: 1)
allow for the
recovery of animal body weight which is reduced following surgery, 2) allow
complete
healing of superficial surgical sites, and) "bulbectomy syndrome" develops
during the first
2 weeks postsurgery.
[00494] Two weeks after surgery, OBX and sham-operated animals are subdivided
into
one of four experimental conditions. One group of OBX animals is administered
daily
injections of saline solution (n = 6 for each surgical condition) or compound
disclosed
herein (1 mg/kg; oral gavage) (n = 6 for each surgical condition). These
groups are included
to examine the effect of chronic administration of compound disclosed herein
(PAK
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inhibitor) on olfactory bulbectomized animals (2 weeks postsurgical recovery +
2 weeks
PAK inhibitor treatment). Administration of the drug or control solution are
given at the
same time each day and in the home cage of each animal. Groups of OBX and sham-
operated animals receive no treatment during this 2-week period and serve as
unhandled
controls. These groups are necessary to examine the persistence of observed
effects of OBX
on dendritic spine density (4 weeks postsurgery). Animals receiving
postsurgery drug
treatment are sacrificed 24 h after the last injection.
[00495] Animals are perfused transcardially with 4% formaldehyde (in 0.1 M
sodium
phosphate buffer, pH = 7.4) under deep anesthesia with sodium pentobarbital
(60 mg/kg) at
the completion of experimental procedures. Following fixation, brains are
removed and
placed in 4% formaldehyde (freshly depolymerized from para-formaldehyde)
overnight.
Brains are then sectioned at 100 m on a vibratome and prepared for Golgi
impregnation
using a protocol adapted from previously described methods (Izzo et al, 1987).
In brief,
tissue sections are postfixed in 1% Os04 for 30 min and then washed in 0.1 M
phosphate
buffer (3 X 15 min). Sections are free-floated in 3.5% K2Cr2O7 solution for 90
min,
mounted between two microscope slides in a "sandwich" assembly, and rapidly
immersed in
a 1% AgN03 solution. The following day, sections are rinsed in ddH 20,
dehydrated in
70% and 100% ethanol, cleared with HistoclearTM, and mounted on microscope
slides with
DPX.
[00496] Dendritic spines are counted on 1250X camera lucida images that
include all
spines observable in each focal plane occupied by the dendrite. Cells are
analyzed only if
they are fully impregnated (CAI: primary apical dendrites extended into
stratum lacunosum
moleculare and basilar dendrites extended into stratum oriens; CA3: primary
apical
dendrites extended into stratum lacunosum moleculare and basilar dendrites
extended into
stratum oriens; dentate gyros: secondary dendrites extended from primary
dendrite within
the molecular layer), intact, and occurring in regions of the section that are
free of blood
vessels, precipitate, and/or other imperfections. Dendritic spines are counted
along the
entire length of secondary oblique dendritic processes (50-100 m) extending
from the
primary apical dendrite within stratum radiatum of area CAI and CA3. In CAI
and CA3,
secondary dendrites are defined as those branches projecting directly from the
primary
apical dendrite exclusive of tertiary daughter branches. In addition, spines
are counted along
the length of secondary dendrites of granule cells in the dentate gyros to
determine if effects
are limited to CAI and CA3. In dentate gyros, secondary dendrites are analyzed
in the
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glutamatergic entorhinal input zone in the outer two-thirds of the molecular
layer.
Approximately 20 dendritic segments (10 in each cerebral hemisphere; 50-100 m
in
length) in each hippocampal subregion (CA 1, CA3, and dentate gyrus) are
examined for
each experimental animal. Treatment conditions are coded throughout the entire
process of
cell identification, spine counting, dendritic length analysis, and subsequent
data analysis.
Analysis of variance and Tukey post-hoc pairwise comparisons are used to
assess
differences between experimental groups.
[00497] When significant changes in dendritic spine density are observed,
camera lucida
images and the Zeiss CLSM measurement program are used to quantify the number
and
length of secondary dendrites. This analysis is necessary as apparent changes
in dendritic
spine density can result from an increase or decrease in the length of
dendrites and not the
formation or loss of spines per se. Photomicrographs are obtained with a
helium-neon 633
laser and Zeiss 410 confocal laser scanning microscope.
Example 50 Treatment of Epilepsy by Administration of a PAK Inhibitor Compound
Disclosed Herein in an Animal Model
[00498] A rat tetanus toxin model of epilepsy is used to evaluate treatment of
epilepsy
with compound disclosed herein.
[00499] Wistar rat pups (Harlan Sprague Dawley, Indianapolis, IN), 10 d of
age, are
anesthetized with an intraperitoneal injection of ketamine and xylazine (33
and 1.5 mg/kg,
respectively). When necessary, this is supplemented by inhalation of
methoxyflurane
(Metofane). Tetanus toxin solution to be injected is generated by dissolving
2.5 or 5 ng of
tetanus toxin in 20 or 40 nl of sterile saline solution. Afterwards, the
tetanus toxin solution
is coinjected into the right hippocampus along with a solution of a compound
disclosed
herein.
[00500] To inject tetanus toxin and a compound disclosed herein, the pups are
placed in
an infant rat stereotaxic head holder, a midline incision is made, and a small
hole is drilled
in the skull. The stereotaxic coordinates for injection are: anteroposterior, -
2.1 mm;
mediolateral, 3.0 mm from the bregma; and dorsoventral, -2.95 mm from the
dural surface.
The toxin and a compound disclosed herein are slowly injected at 4 nl/min.
After injection,
the needle is left in place for 15 min to reduce reflux up the needle track.
During injections,
the body temperature of rat pups is maintained by a warmed (electrically
regulated) metal
plate. Littermates, stereotaxically injected with sterile saline, or untreated
rats serve as
controls.
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[00501] The frequency of behavioral seizures is monitored for 1 hr/day for 10
consecutive days after tetanus toxin/the test compound injections. The types
and duration of
seizures are scored. Wild running seizures are most easily identified.
[00502] After seizure scoring on the 10th day animals are perfused
transcardially and
dendritic spines in the CA3 region are counted and analyzed as described
above.
[00503] The t test for comparison of two independent means is used in
comparing the
number of seizures in treated vs. untreated rats and in comparing dendritic
and axon arbors
in experimental and control rats. When data are not normally distributed, a
Mann-Whitney
U test is used. Sigma Stat is used to perform all statistical tests. It is
expected that treatment
with a compound disclosed herein will reduce the frequency and severity of
seizures.
Example 51 Treatment of Mild Cognitive Impairment by Administration of a PAK
Inhibitor in an Animal Model
[00504] The ability of a compound of Formula I-XV to delay or halt the
progression of
symptoms of Mild Cognitive Impairment (i.e., their mouse analogs) is tested in
a Tg2576
mouse model of Mild Cognitive Impairment (Young et al. (2009), Neurobiology
ofAging,
30:1430-1443).
[00505] Thirty-two Tg2576 male mice (ages 3-4 months) and their wild-type
littermates
(n=8) are divided into a treatment groups (1 mg/kg oral gavage), placebo
groups (0.1%
DMSO in physiological saline solution) and wild-type and analyzed for
behavioral
differences in olfactory discrimination and odor recognition memory using a
mouse odor
span task apparatus (Young et al. (2007), Neuropharmacology 52:3634-645).
[00506] In each mouse odor span task test, a mouse is placed on an elevated
wooden
platform (61 cm x 61 cm) using numbers as location identifiers. Numbers 1-24
are used,
with 1, 7, 13, and 19 at each corner and the intervening five numbers evenly
spaced between
the corners locations. The following odors are used: allspice, Chinese five
spice, cinnamon,
nutmeg, coriander, fenugreek, ginger, paprika, thyme, parsley, dill, oregano,
sage, mint,
rosemary, onion powder, caraway seed, celery salt, cocoa, coffee powder
(Maxwell
House ), and English breakfast tea (Twinnings ). All scented mixtures are
created by
adding 3 g of a specific odor to 100 g of woodchip and 18 crushed food pellets
(Noyes
Precision Pellets, Lancaster, UK). These mixtures are placed in white
porcelain bowls (5.5
cm in diameter, 3.5 cm high; Fisher Loughborough, UK) and are marked with a
letter of the
alphabet (A-v) identifying the odor.
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[00507] After the mice are introduced to each odor, the odor span task tests
are
habituated to the testing protocol. Habituation is conducted as follows: Span
0: a bowl is
baited and placed on the platform at the chosen location; with the
introduction of the mouse
(which always faces the experimenter's left; location 16) a timer is started.
Digging in the
bowl for the food pellet (reward) stops the timer and the mouse is required to
remember the
odor in that bowl. Following consumption of the reward, the mouse is removed
to a clear
Perspex cage located below the platform, a new bowl and location is selected,
the bowl is
baited and placed appropriately. The first bowl (no longer baited) is moved to
a new
location. Span 1: the mouse is placed back on the platform and the timer is
restarted, with
the mouse required to dig only in the novel bowl. After digging in either bowl
the timer is
stopped, and if a correct choice is made, the mouse is given time to consume
the reward
before being returned to the clear cage. The accuracy of this span is noted,
for once the non-
match rule is acquired this gave an indication of the ability of the mouse to
perform a simple
two-odor discrimination. Span 2: a third (baited) bowl is then placed on the
platform in the
designated location and the two previously sampled bowls are repositioned as
required. If
an incorrect response is made (digging in a previously sampled bowl), the
three bowls are
randomly relocated and the span is repeated until a correct response is made.
The span
number is then increased with every correct response until span 21 (22 bowls)
is completed
or the mouse has spent 10 min on the platform. Any incorrect response will
lead to a
repetition of that span with all bowls being randomly relocated.
[00508] The number of odors (bowls) a mouse remembers prior to erring is
regarded as
the mouse's span length for that session. The total number of spans completed
is also
recorded as are errors per session and % accuracy [(spans completed/spans
completed +
errors) x 100]. Each subject's mean span latency (total correct latency/spans
completed) is
also calculated, with time to first sample (latency to complete span 0) being
recorded to
ensure that mice takes a comparable amount of time to engage in the task. A
bowl is
randomly selected every third span (spans 2, 5, 8 and 11) and replaced with an
identical yet
previously non-sampled odor filled bowl, which will unmask any scent marking
strategy. In
addition, between every session the table is wiped down with ethanol. The mice
are
continuously trained until a stable level of performance is reached, with
performance then
being assessed over 4 consecutive days.
[00509] The odor span task test is conducted at 4 months, 8 months and 12
months to
evaluate the progression of Mild Cognitive Impairment in the Tg2576 mice. In
this test, a
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significant increase in Span Length, a significant increase in % Accuracy, or
significant
decrease in errors per session over the course of the experimental period
(e.g., results at 4
month vs. 8 months, results at 4 month vs. 8 months) in the test compound
groups relative
to the placebo group (and/or as compare to the wild-type group) is indicative
of a successful
treatment effect.
[00510] Statistical Analysis. Statistical analysis is performed by ANOVA or
repeated
ANOVA. Differences between groups are considered significant at p < 0.05.
Example 52 Treatment of Mild Cognitive Impairment by Administration of a PAK
Inhibitor in an Animal Model
l0 [00511] The ability of compounds of Formula I-V to delay or halt the
progression of
behavioral symptoms and anatomical symptoms of Mild Cognitive Impairment
(i.e., their
mouse analogs) is tested in a Mo/Hu APP695swe mouse model of Alzheimer's
disease
(Knafo et al (2007), Cerebral Cortex Advance Access, July 28, 2008).
[00512] Forty Mo/Hu APP695swe mice (ages 3 months) are divided into treatment
groups (1 mg/kg oral gavage) and a placebo group (0.1% DMSO in physiological
saline
solution) and analyzed for memory differences in open field, prepulse
inhibition, and hidden
food behavioral tests, with an interval of about one week between each type of
test. Each
series of open field, prepulse inhibition, and hidden food behavioral tests
are conducted at 3
months, 6 months, 9 months, and 12 months to evaluate the progression of
cognitive
impairment in the APP695swe mice.
[00513] In the open field test, each mouse is placed in a novel open field box
(40 cm X
40 cm; San Diego Instruments, San Diego, CA) for two hours. Horizontal and
vertical
locomotor activities in the periphery as well as the center area are
automatically recorded by
an infrared activity monitor (San Diego Instruments). Single breaks are
reported as
"counts." In this behavioral test, a significant reduction in total activity
in the test groups
relative to the placebo group over the course of the testing period indicates
a possible
treatment effect.
[00514] In the hidden food test, mice are food-deprived for 24 h. After
habituation to a
new cage for 5 min, a food pellet is hidden under the cage bedding. The time
it takes for the
mouse to find the food pellet is measured until a maximum of 10 min is
reached. In this
behavioral test, a significant reduction in time to find the food pellet in
the test groups
relative to the placebo group over the course of the testing period is
indicative of a
successful treatment effect.
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[00515] In the Morris Water Maze test, mice are placed in a pool with an exit
platform.
When released, the mouse swims around the pool in search of an exit while
various
parameters are recorded, including the time spent in each quadrant of the
pool, the time
taken to reach the platform (latency), and total distance traveled. The
animal's ability to
quickly find the platform, and on subsequent trials (with the platform in the
same position)
the ability to locate the platform more rapidly is recorded. Any significant
showing of a
reduced progression of the decline in performance in the test groups relative
to the placebo
group over the course of the testing period is indicative of a successful
treatment effect.
[00516] The radial arm maze test, measures spatial learning and memory in
mice. Mice
are placed in an apparatus comprising eight equidistantly-spaced arms, each
about 4 feet
long, and all radiating from a small circular central platform. Food is placed
at the end of
each arm. The design ensures that, after checking for food at the end of each
arm, the mouse
is always forced to return to the central platform before making another
choice. The ability
of mice to remember locations on the arm is measured to determine memory and
spatial
learning. A significant showing of reduced progression in the decline of
performance in the
test groups relative to the placebo group over the course of the testing
period is indicative of
a successful treatment effect.
[00517] The T-maze is designed to test spatial working memory to assess
hippocampal
and forebrain function. In the "delayed non-match to place" or "delayed
alternation" test,
there are 2 runs per trial. On the first, or sample run, the mouse is placed
in the start arm of
the T-maze and allowed to enter a goal arm. The mouse is then removed from the
maze for
a specified delay period. After the delay, the mouse is returned for the
choice run. The
choice of arm used by the mouse is scored according to variety of criterion,
including
spontaneous alternation, cued reward, or to indicate a preference. Based on
the criterion
used in an experiment, the T-maze can be used to test learning and memory,
preferences for
stimuli or reward, or spontaneous alternation behavior. A significant showing
of reduced
progression in the decline of performance in the test groups relative to the
placebo group
over the course of the testing period is indicative of a successful treatment
effect.
[00518] In the prepulse inhibition test, acoustic startle and prepulse
inhibition responses
are measured in a startle chamber (San Diego Instruments). Each mouse is
individualed to
six sets of seven trail types distributed pseudorandomly: pulse-alone trials,
prepulse-pulse
trials, and no-stimulus trials. The pulse used is 120dB and the prepulse is 74
dB. A
significant showing of reduced progression in the decline of the prepulse
inhibition response
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in the test groups relative to the placebo group over the course of the
testing period is
indicative of a successful treatment effect.
[00519] In the forced swim test, each mouse is put in a large plastic
cylinder, which is
half-filled with room temperature water. The test duration is 6 min, during
which the
swim/immobility times are recorded. In this behavioral test, a significant
showing of
reduced progression in the decline of immobility in the test groups relative
to the placebo
group over the course of the testing period is indicative of a successful
treatment effect.
[00520] In order to evaluate the ability of the test compounds to alter brain
morphology,
an MRI study is conducted on placebo-treated and test compound-treated groups
of Mo/Hu
APP695swe mice. In vivo MRI experiments are performed on an 11.7T Bruker
Biospec
small animal imaging system. A three-dimensional, fast-spin echo, diffusion
weighted
(DW) imaging sequence with twin navigation echoes is used to assess the ratio
of lateral
ventricle volume to total brain volume. A decrease in this ratio in the test
compound-treated
groups relative to the ratio observed in the placebo-group is indicative of a
successful
treatment effect.
[00521] Statistical Analysis. Statistical analysis is performed by ANOVA or
repeated
ANOVA. Differences between groups are considered significant at p < 0.05.
Example 53 Treatment of Autism by Administration of a PAK Inhibitor in an
Animal
Model
[00522] The ability of a compound of Formula I-XV described herein (a PAK
inhibitor)
to alleviate, reduce the severity of, or inhibit the progression of symptoms
of autism (i.e.,
their mouse analogs) is tested in a FMR1 KO mouse model.
[00523] Twenty-four FMR1 KO male mice (age 2 months) are divided into Group 1
(n=6) and Group 2 (n=6) treatment groups (1 mg/kg oral gavage of a compound of
Formula
I-V described herein), a placebo Group (Group 3) (n=6) (0.1% DMSO in
physiological
saline solution) and wild-type (Group 4) (n=6) and are analyzed for behavioral
differences
using the Open Field Test.
[00524] Open Field Test. The mice in Groups 1-4 are subjected to the open
field test
according to standard procedures. Each of the mice ran for 60 minutes in a
VersaMax
activity monitor chamber (Accuscan Instruments). Open field activity is
detected by
photobeam breaks and is analyzed by the VersaMax software. Stereotypy is
recorded when
the mouse breaks the same beam (or set of beams) repeatedly. Stereotypy count
is the
number of beam breaks that occur during this period of stereotypic activity.
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[00525] FMR1 KO mice are known to exhibit three abnormal behaviors compared to
wild-type mice (Peier et., 2000, Hum. Mol. Genet., 9:1145): (i) hyperactivity-
they travel a
longer distance and move for a longer period of time than wild-type; (ii)
stereotypy-they
exhibit a higher number of repetitive behaviors than wild-type; and (iii) hypo-
anxiety-they
stay in the center field for a longer period of time and in the corners of the
field for shorter
periods of time than wild-type.
[00526] It is expected that the FMR1 mice in treatment Group 1 and treatment
Group 2
will perform comparable to the wild-type controls (Group 4) for: (i)
hyperactivity; (ii)
stereotypy; and (iii) hypo-anxiety as measured in the Open Field Test, whereas
the FMR1
mice in Group 3 will exhibit abnormal behavior. This indicates that treatment
of FMR1 KO
mice with PAK inhibitors of a compound of Formula I-XV described herein
restores
activity, repetitive behavior, and anxiety to wild-type levels.
[00527] Statistical Analysis. Statistical analysis is performed by ANOVA or
repeated
ANOVA. Differences between groups are considered significant at p < 0.05.
Example 54 Treatment of Autism by Administration of a PAK Inhibitor in an
Animal
Model
[00528] The ability of a compound of Formula I-XV described herein (a PAK
inhibitor)
to delay or halt the progression of behavioral symptoms of autism (i.e., their
mouse analogs)
is tested in a BTBR T1tfJ mouse model of autism syndrome (McFarlane et al.,
Genes, brain,
and behavior (2007)).
[00529] BTBR T1tfJ is an inbred mouse strain that shows robust behavioral
phenotypes
with analogies to all three of the diagnostic symptoms of autism, including
well-replicated
deficits in reciprocal social interactions and social approach, unusual
patterns of ultrasonic
vocalization, and high levels of repetitive self-grooming.
[00530] Twenty BTBR T1tfJ male mice (age 2 months) are divided into Group 1
(n=5)
and Group 2 (n=5) treatment groups (1 mg/kg oral gavage of a compound of
Formula I-V
described herein), a placebo Group (Group 3) (n=5) (0.1% DMSO in physiological
saline
solution) and wild-type (Group 4) (n=5) and are analyzed for behavioral
differences using
the sociability test and self grooming test described below.
[00531] Sociability Test. Social approach behaviors are tested in an automated
3-
chambered apparatus using methods similar to those previously described (Moy
et al., 2004;
Nadler et al., 2004; Crawley et al., 2007; McFarlane et al., 2007; Moy et al.,
2007). Briefly,
the apparatus is a rectangular, three-chambered box made from clear
polycarbonate.
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Retractable doorways built in the two dividing walls allow access to the side
chambers.
Quantification of entries and duration in the chambers is automatically
measured by
photocells embedded in the doorways. The apparatus is cleaned with 70% ethanol
and water
between subjects.
[00532] Animals to be used as "strangers" are male 129Sv/ImJ and AJ mice, aged
8-14
weeks old (The Jackson Laboratory (Bar Harbor, ME)). Strangers are habituated
to the
apparatus and to the wire cup enclosure before the start of experiments, for
10 min per day
for three consecutive days. The subject mouse is allowed to acclimate to the
apparatus for
20 min before the sociability test, 10 min in the central chamber with the
doors closed and
another 10 min in the entire empty arena with the doors open. The subject is
then briefly
confined to the center chamber while a novel object (inverted wire cup, Galaxy
Cup) is
introduced into one of the side chambers. A stranger mouse enclosed in an
identical wire
cup is placed in the other side chamber. An upright plastic drinking cup, held
in place by a
lead weight in the cup, is placed on the top of each inverted wire cup to
prevent the subject
from climbing onto the top of the wire cup. The location for the novel object
and the
stranger mouse alternates between the left and right chambers across subjects.
After both
stimuli are positioned, the doors are simultaneously re-opened and the subject
is allowed
access to all three chambers for 10 min. Measures to be taken include time
spent in each
chamber, time spent sniffing each cup, and number of entries. An observer
uninformed of
the genotypes scores time spent sniffing with a stopwatch.
[00533] Self-Grooming. The test is performed as previously described
(McFarlane et al.,
2007). Each subject is placed individually in a clean standard mouse cage and
allowed to
acclimate for 10 min. Following this habituation period, subjects are observed
for another
10 min, during which time cumulative time spent in self-grooming is scored by
an
experimenter sitting approximately 2 meters from the test cage. A silenced
stopwatch is
used for scoring cumulative time spent grooming during the 10 min test
session.
[00534] It is expected that the BTBR T1tfJ mice in treatment Group 1 and
treatment
Group 2 will perform comparable to the wild-type controls (Group 4) for: (i)
sociability and
(ii) self-grooming, whereas the BTBR T1tfJ mice in Group 3 will exhibit
abnormal
behavior. This indicates that treatment of BTBR T1tfJ mice with PAK inhibitors
of a
compound of Formula I-XV described herein restores low sociability and
repetitive self-
grooming behavior to wild-type levels.
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[00535] Statistical Analysis. Statistical analysis is performed by ANOVA or
repeated
ANOVA. Differences between groups are considered significant at p < 0.05.
Example 55 Treatment of Learning Deficits Associated with Neurofibromatosis
Type 1
by Administration of a PAK Inhibitor in an Animal Model
[00536] Neurofibromatosis Type 1 (NF 1) is one of the most common single-gene
disorders that causes learning deficits in humans. Mice carrying a
heterozygous null
mutation of the Nfl gene (Nfl+i-) show important features of the learning
deficits associated
with NF 1.
[00537] Generation of different genetically modified mice are described in
Johnson, L.K-
r. et al., Genes Dev. 11, 2468-81 (1997); Jacks, T. et al., Nature Genet. 7,
353-61 (1994);
and Umanoff, H., Edelmann, W., Pellicer, A. & Kucherlapati, R., Proc. Natl.
Acad. Sci.
USA, 92, 1709-13 (1995).
[00538] Water Maze Experiment: The protocol for the water maze experiment is
described in Costa, R.M. et al., Nature Genet. 27, 3 99-405 (2001). Mice from
the are given
two trials per day (30-s intertrial intervals) with a probe trial (60s) at the
end of training
day7. In the probe trial, WT mice spent significantly more time in the
training quadrant
compared to Nf1+i- animals. The PAK inhibitor test compound is dissolved in
sterile saline
solution and injected every day for several days (typical dosing regimen are 2
to 5 days of
dosing). The Water Maze experiment is performed between 2 and 8 hours
following the
final dose.
[00539] Electrophysiology: For field potentials, recordings are made from
transverse
hippocampal slices (400 pm thick) in a submerged recording chamber perfused (2
ml min)
with artificial cerebrospinal fluid (ACSF) containing (in mM): 120 NaCl, 3.5
KC1, 2.5
CaC12, 1.3 Mg2SO4, 1.25 NaH2PO4, 26 NaHC03, and 10 D-glucose at 30 deg. C
(saturated
with 95% 02 and 5% C02). For LTP experiments, EPSPs are evoked alternatively
in
separate pathways (control and tetanized) in a CA1 Schaffer
collateral/commissural
afferents with 100- s test pulses through two stimulating electrodes (about
300 mm from
the Pt/Ir recording electrode. The stimulation strength in both stimulating
electrodes is set
to 60 A. After a 10-min baseline period, LTP is induced in one pathway
according to a
HFS or TBS protocol. The amount of potentiation is calculated as a percentage
of the
baseline EPSP slope.
[00540] To access inhibition in Nf1+i mice, IPSPs from CAI pyramidal neurons
are
measured using whole-cell (blind technique) bridge mode recordings (Axoclamp
2B, Axon
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Instruments). IPSPs are evoked through a stimulating electrode placed in the
Schaffer
collateral/commissural afferents from applying different stimulation strengths
(from 10 to
100 A in steps of 10 A). The IPSP amplitude is measured with five IPSPs
averaged for
each neuron per stimulation strength. The intracellular solution contains (in
mM): 135
potassium gluconate, 5 HEPES, 2 Mg2+ -ATP, 5 MgC12, 0.3 GTP, 0.05 EGTA. To
evoke
IPSPs monosynaptically, AP5 and CNQX (10 M) are present in the ACSF.
[00541] Statistical Analysis: Acquisition data from the water maze are
analyzed by
repeated-measures ANOVA. Per cent time in training quadrant for the different
genotypes
are analyzed using single factor ANOVA; post-hoc comparisons between genotypes
are
carried out when appropriate. Planned comparisons using a paired t-test are
used to analyze
the proximity data. LTP is analyzed using single factor ANOVA on the average
amount of
LTP 30-40 min after induction. Inhibition and input-output curves are analyzed
using
ANOVA and post-hoc comparisons are performed when appropriate.
Example 56 Growth Inhibition of a Compound of Formula I in Various Cancer Cell
Lines
Methodology: 60 cell lines (CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR,
A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-
H522, COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW-620, SF-268, SF-
295, SF-539, SNB-19, SNB-75, U251, LOX IMV1, MALME-3M, M14, SK-MEL-2, SK-
MEL-28, SK-MEL-5, UACC-257, UACC-62, IGR-OV1, OVCAR-3, OVCAR-4, OVCAR-
5, OVCAR-8, SK-OV-3, 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10, UO-31,
PC-3, DU-145, MCF7, NCI/ADR-RES, MDA-MB-231, HS 578T, MDA-MB-435, MDA-
MB-468, BT-549, and T-47D) are grown in RPMI-1640 medium with 10% FBS. Stock
solutions of a test compound are prepared in DMSO. Concentrations of from
about 0.001
pM to about 20 pM of each compound in RPM- 1640 media are prepared. The test
compound is added to wells containing 50 pL of cells and medium. A CellTiter-
Glo (CTG)
assay is carried out on the 0 hr plate to obtain a 0 hr count. Cells are
exposed to the test
compound for 72 hours. Following the exposure period, the plates are assayed
using CTG.
Luminescence is recorded on Synergy. A test compound described herein
exhibited a G150
in various cell lines of less than about 1 pM.
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Example 57 Clinical Trial: Treatment of Schizophrenia with a PAK Inhibitor
Compound Disclosed Herein
[00542] The following human clinical trial is performed to determine the
safety and
efficacy of a PAK inhibitor compound for the treatment of schizophrenia.
[00543] Sixty patients are recruited via referrals from community mental
health teams,
after the patients have been diagnosed with schizophrenia using the Structured
Clinical
Interview for DSM-IV ("SCID"; First et al., (1995), Structured Clinical
Interview for DSM-
IVAxis I Disorders, Patient Edition (SCID-P), version 2, New York State
Psychiatric
Institute, Biometrics Research, New York).
[00544] A screening visit is arranged and a full explanation of the study
prior to
screening is provided if the patient appeared suitable for and interested in
taking part. For
inclusion, all patients are required to meet the following criteria: (i) aged
between 18 and 60
years, (ii) receiving stable treatment with an atypical (Risperidone,
Olanzapine, Quetiapine)
antipsychotic and have stable psychotic symptoms (i.e. no change in
medication/dose of
current medication over last 6 weeks and unlikely to require change in
antipsychotic
medication), (iii) negative urine screening for illicit drugs and negative
pregnancy test for
female patients, (iv) cooperative, able to ingest oral medication and willing
to undertake
repeated cognitive testing, (v) able to provide written informed consent, (vi)
reading ability
of not more than 40 errors on the National Adult Reading (Nelson et al,
(1991)), and (vii)
between 1 and 2 standard deviations (S.D.) below expected performance on the
basis of age
and education level on the California Verbal Learning Test (Delis et al.,
1987). In addition,
the following criteria are used to define unsuitable patients: (i) concurrent
DSM-IV
diagnosis, (ii) current treatment with benzodiazepines or antidepressants,
(iii) history of
neurodegenerative disorder in first degree relative (e.g. AD, Parkinson's
disease,
Huntington's disease, multiple sclerosis), (iv) history of DSM-IV substance
dependence in
the last year or substance abuse within last month, (v) lifetime history of
trauma resulting in
loss of consciousness for 1 h or longer, (vi) participation in another
investigational drug trial
within 6 weeks prior to study entry, (vii) recent (within last 3 months)
history of suicidal or
violent acts, and (viii) current diagnosis of uncontrollable seizure disorder,
active peptic
ulceration, severe and unstable cardiovascular disease or/and acute severe
unstable asthma.
The study procedures are approved by an institutional ethics review board. All
patients in
the study must provide written informed consent.
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[00545] After screening has identified suitable patients that have provided
informed
consent, patients are placed on a single-blind placebo for 1 week. After 1
week on placebo
(baseline), all patients complete a comprehensive cognitive test battery and
undergo clinical
assessments, and then are randomized into a double-blind protocol so that,
half of the
sample received a compound disclosed herein capsules and the remaining half
received
placebo for the next 24 weeks. Cognitive and clinical assessments are carried
out again at
12 weeks and 24 weeks.
[00546] Patients assigned to the treatment group will receive 1.5 mg twice a
day for the
first 2 weeks, 3 mg twice a day over the next 2 weeks, 4.5 mg twice a day dose
for the next
2 weeks and then 6 mg twice a day for the remaining period so at the time of
12 weeks
cognitive assessments all patients are on the maximum dose. The placebo group
will receive
identical appearing capsules containing ascorbic acid (100 mg).
[00547] Symptoms are rated within 4 days of cognitive testing using the
Positive and
Negative Syndrome scale (PANSS) (Kay et al. (1987), Schizophr Res, 13:261-276)
on all
three occasions. Side effects are also assessed within 4 days of testing using
the Abnormal
Involuntary Movement Scale (AIMS) (Guy, (1976), ECCDEU Assessment Manualfor
Psychopharmacology (revised), DHEW Publication No. (ADM)National Institutes of
Mental Health, Rockville, MD, pages 76-338). Inter-rater reliability is
carried out for
PANSS at 6 monthly intervals by rating exemplar cases based on patient
interviews on
videotapes.
[00548] The cognitive battery includes measures of executive functioning,
verbal skills,
verbal and spatial working memory, attention and psychomotor speed. The
battery is
administered to all patients on all three occasions in the same fixed order
(e.g., MATRICS
cognitive battery, BACS score, and performance in Wisconsin Card Sort Test).
Patients are
allowed to take breaks as needed in order to obtain maximal performance at all
times. Tests
are administered and scored by trained psychologists who are blind to
patients' group
affiliations and are not involved in patients' treatment plan in any way.
[00549] Patients are told that the aim of the study is to investigate the
cognitive effects of
a compound disclosed herein. They are requested to abstain from alcohol for at
least 24 h
prior to their scheduled cognitive testing.
[00550] The patients in the treatment and placebo groups are compared on
demographic,
clinical, and cognitive variables obtained at baseline using independent
sample I-tests.
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[00551] The effects of the test compound on positive symptoms, negative
symptoms,
general psychopathology score, total PANSS scores, and the scores on the AIMS
are
analyzed (separately) by 2 (Treatment, placebo) x 3 (Time: baseline, 12 weeks,
24 weeks)
analysis of variance (ANOVA).
[00552] All cognitive variables are first examined for their distribution
properties, i.e., to
ensure normality. The cognitive effects of the test compound over time are
then evaluated
by Treatment x Time ANOVA, performed separately for each variable, with Time
as a
within-individuals factor and Treatment as a between-individuals factor,
followed by post-
hoc mean comparisons wherever appropriate. All cognitive effects are then re-
evaluated
using ANOVA performed separately on change scores computed for each variable
(12
weeks data minus baseline data, 24 weeks data minus baseline data). Alpha
level for testing
significance of effects is p = 0.05.
Example 58 Clinical Trial: Treatment of Epilepsy with a PAKI/PAK3 Inhibitor
[00553] This is a 24-week study of an oral PAKI/PAK3 inhibitor in symptomatic
patients with a diagnosis of epilepsy. This is an open-label, single-arm study
to evaluate the
dosing, tolerability, effectiveness and safety of a PAKI/PAK3 inhibitor as
initial therapy for
epilepsy. A total of 30 subjects will enrolled in the study.
[00554] Study Type: Interventional
Primary Outcome Measures:
[00555] Comparison of the mean stabilized dose of a PAKI/PAK3 inhibitor during
the
last 28 days of treatment between patients reporting 1 to 3 seizures versus
patients reporting
more than 3 seizures, during the 3 months prior to study entry
Secondary Outcome Measures:
[00556] Influence of other patient characteristics on dose; Proportion of
subjects
remaining seizure-free; Time to stabilized dose; Reduction in seizure
frequency.
Inclusion Criteria:
[00557] Subjects having new-onset epilepsy or epilepsy relapse characterized
by partial-
onset seizures or primary generalized tonic-clonic seizures; Having at least 1
seizure within
the 3 months prior to entry; Subjects who are previously untreated for
epilepsy, previously
treated for epilepsy, or if currently taking epilepsy medication, must have
been taking it for
less than 6 weeks
Exclusion Criteria:
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[00558] Subjects currently on any medication for epilepsy for greater than 6
weeks;
Having active liver disease.
Experimental Design
[00559] Patients are divided into two groups, a placebo group and a PAK1/PAK3
inhibitor group. Patients are administered tablets starting at 50 milligrams
per day and
titrated to an individualized optimal dose, up to a maximum of 400 milligrams
per day of
the PAK1/PAK3 inhibitor by the end of week 6. Patients will take tablets by
mouth twice a
day (morning and evening) for 24 weeks. Changes to this schedule will be based
on a risk-
benefit assessment of the patient's clinical condition by the investigator,
such as tolerability,
or reaching a stable dose sufficient to control their seizures.
[00560] Patients are evaluated at weekly visits over a period of 6 weeks.
Groups are
compared using ANOVA. Single variable differences are analyzed using an
independent
samples t-test. A Pearson's coefficient is used to determine relationship
between seizure
frequency and medication dose.
Example 59 Clinical Trial: Treatment of Alzheimer's disease with a PAK
Inhibitor
[00561] The following human clinical trial is performed to determine the
safety and
efficacy of the PAK inhibitor disclosed herein for the treatment of
Alzheimer's disease. The
study aims to provide preliminary estimates of effect of administration of a
PAK inhibitor in
delaying progression of disease over a study period of one year.
[00562] Sixty patients between the ages of 55 and 80 are recruited via
referrals from
hospitals, after the patients have been diagnosed with mid stage Alzheimer's
disease using
the Mini-Mental State Exam scores and a clinical interview.
[00563] A screening visit is arranged and a full explanation of the study
prior to
screening is provided if the patient appeared suitable for and interested in
taking part. For
inclusion, all patients are required to meet the following criteria: (i)
diagnosis of
Alzheimer's disease (ii) a study partner who can attend all study visits (iii)
negative urine
screening for illicit drugs (iv) cooperative, able to ingest oral medication
and willing to
undertake repeated cognitive testing, (v) able to provide written informed
consent.
Exclusion criteria include (i) significant neurological disease other than
Alzheimer's disease
(ii) significant depression or other psychiatric disorder (iii) unstable
medical conditions. The
study procedures are approved by an institutional ethics review board. All
patients in the
study must provide written informed consent.
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[00564] After screening has identified suitable patients that have provided
informed
consent, patients are placed on a single-blind placebo for 1 week. After 1
week on placebo
(baseline), all patients complete a comprehensive cognitive test battery and
undergo clinical
assessments, and then are randomized into a double-blind protocol so that,
half of the
sample received test compound capsules and the remaining half received placebo
for the
next 52 weeks. Cognitive and clinical assessments are carried out again at 12
weeks, 26
weeks and 52 weeks.
[00565] Patients assigned to the test compound group will receive a dose twice
a day for
12 weeks at increasing doses. Cognitive assessments for all patients are on
the maximum
dose. The placebo group will receive identical appearing capsules containing
ascorbic acid
(100 mg).
[00566] The cognitive battery includes measures of executive functioning,
verbal skills,
verbal and spatial working memory, attention and psychomotor speed. The
battery is
administered to all patients on all three occasions in the same fixed order
(e.g., Mini-Mental
State Examination (MMSE), MATRICS cognitive battery, BACS score, and
Alzheimer's
disease Assessment Scale - Cognitive Subscale (ADAS-Cog)). Patients are
allowed to take
breaks as needed in order to obtain maximal performance at all times. Tests
are
administered and scored by trained psychologists who are blind to patients'
group
affiliations and are not involved in patients' treatment plan in any way.
Alzheimer's disease
Cooperative Study - Activities of Daily Living (ADCS-ADL) is also recorded.
[00567] Patients are told that the aim of the study is to investigate the
cognitive effects of
the test compound. They are requested to abstain from alcohol for at least 24
h prior to their
scheduled cognitive testing.
[00568] The patients in the test compound and placebo groups are compared on
demographic, clinical, and cognitive variables obtained at baseline using
independent
sample I-tests.
[00569] The effects of the test compound on Neuropsychological Test Battery
and
Neuropsychiatric Inventory (NPI) are analyzed (separately) by 2 (Treatment:
Test
compound, placebo) x 3 (Time: baseline, 12 weeks, 26 weeks, 52 weeks) analysis
of
variance (ANOVA).
[00570] All cognitive variables are first examined for their distribution
properties, i.e., to
ensure normality. The cognitive effects of test compound over time are then
evaluated by
Treatment x Time ANOVA, performed separately for each variable, with Time as a
within-
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individuals factor and Treatment as a between-individuals factor, followed by
post-hoc
mean comparisons wherever appropriate. All cognitive effects are then re-
evaluated using
ANOVA performed separately on change scores computed for each variable (12
weeks data
minus baseline data, 26 weeks, 52 weeks data minus baseline data). Alpha level
for testing
significance of effects is p = 0.05.
[00571] Primary outcome measure is an improvement in (ADAS-Cog) scores.
Secondary
outcome measures are improvement in (MMSE) scores and (ADCS-ADL).
Example 60 Clinical Trial: Treatment of Mild Cognitive Impairment with a PAK
Inhibitor
l0 [00572] The following human clinical trial is performed to determine the
safety and
efficacy of the PAK inhibitor having the structure of Formula I-XV for the
treatment of
Mild Cognitive Impairment. The study aims to provide preliminary estimates of
effect of
administration of a PAK inhibitor in delaying progression of the disease over
a study period
of one year.
[00573] Sixty patients between the ages of 45 and 80 are recruited via
referrals from
hospitals, after the patients have been diagnosed with Mild Cognitive
Impairment using the
Mini-Mental State Exam scores (MMSE score of 21-24) and a clinical interview.
[00574] A screening visit is arranged and a full explanation of the study
prior to
screening is provided if the patient appeared suitable for and interested in
taking part. For
inclusion, all patients are required to meet the following criteria: (i)
diagnosis of Mild
Cognitive Impairment (ii) a study partner who can attend all study visits
(iii) negative urine
screening for illicit drugs (iv) cooperative, able to ingest oral medication
and willing to
undertake repeated cognitive testing, (v) able to provide written informed
consent.
Exclusion criteria include (i) significant neurological disease and/or
dementia (including
Alzheimer's disease) (ii) significant depression or other psychiatric disorder
(iii) unstable
medical conditions. The study procedures are approved by an institutional
ethics review
board. All patients in the study must provide written informed consent.
[00575] After screening has identified suitable patients that have provided
informed
consent, patients are placed on a single-blind placebo for 1 week. After 1
week on placebo
(baseline), all patients complete a comprehensive cognitive test battery and
undergo clinical
assessments, and then are randomized into a double-blind protocol so that,
half of the
sample received test compound capsules and the remaining half received placebo
for the
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next 52 weeks. Cognitive and clinical assessments are carried out again at 12
weeks, 26
weeks and 52 weeks.
[00576] Patients assigned to the test compound group will receive 1.5 mg twice
a day for
the first 2 weeks, 3 mg twice a day over the next 2 weeks, 4.5 mg twice a day
dose for the
next 2 weeks and then 6 mg twice a day for the remaining period so at the time
of 12 weeks
cognitive assessments all patients are on the maximum dose. The placebo group
will receive
identical appearing capsules containing ascorbic acid (100 mg).
[00577] The cognitive battery includes measures of executive functioning,
verbal skills,
verbal and spatial working memory, attention and psychomotor speed. The
battery is
administered to all patients on all three occasions in the same fixed order
(e.g., Mini-Mental
State Exam (MMSE), Wechsler Intelligence Scale, Wechsler Memory Scale,
Dementia
Rating Scale (DRS) or Auditory Verbal Learning Test (AVLT)). Patients are
allowed to
take breaks as needed in order to obtain maximal performance at all times.
Tests are
administered and scored by trained psychologists who are blind to patients'
group
affiliations and are not involved in patients' treatment plan in any way.
[00578] Patients are told that the aim of the study is to investigate the
cognitive effects of
a compound of Formula I-XV. They are requested to abstain from alcohol for at
least 24 h
prior to their scheduled cognitive testing.
[00579] The patients in the test compound group and placebo groups are
compared on
demographic, clinical, and cognitive variables obtained at baseline using
independent
sample I-tests.
[00580] The effects of test compound on Neuropsychological Test Battery and
Neuropsychiatric Inventory (NPI) are analyzed (separately) by 2 (Treatment:
test
compound, placebo) x 3 (Time: baseline, 12 weeks, 26 weeks, 52 weeks) analysis
of
variance (ANOVA).
[00581] All cognitive variables are first examined for their distribution
properties, i.e., to
ensure normality. The cognitive effects of the test compound(s) over time are
then evaluated
by Treatment x Time ANOVA, performed separately for each variable, with Time
as a
within-individuals factor and Treatment as a between-individuals factor,
followed by post-
hoc mean comparisons wherever appropriate. All cognitive effects are then re-
evaluated
using ANOVA performed separately on change scores computed for each variable
(12
weeks data minus baseline data, 26 weeks, 52 weeks data minus baseline data).
Alpha level
for testing significance of effects is p = 0.05.
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[00582] Primary outcome measure is an improvement in MMSE scores. Secondary
outcome measures are improvements in DRS scores and AVLT scores.
Example 61 Clinical Trial: Treatment of Amnestic Mild Cognitive Impairment
with a
Compound of Formula I-XV
[00583] This is a 40-week, randomized, double blind, parallel groups designed,
study of
an oral inhibitor having the structure of Formula I-XV in symptomatic patients
with a
diagnosis of amnestic Mild Cognitive Impairment. This pilot study aims to
provide
preliminary estimates of effect of an inhibitor having the structure of
Formula I-XV on
cognitive deficits and whether the effects differ between amnestic Mild
Cognitive
Impairment patients treated with an inhibitor, and amnestic Mild Cognitive
Impairment
patients treated with donepezil. A total of 30 subjects will enrolled in the
study.
[00584] Study Type: Interventional
[00585] Study Design: Treatment, Randomized, Double Blind (Subject,
Investigator),
Active Control, Parallel Assignment, Efficacy Study
Primary Outcome Measures:
[00586] To provide preliminary estimates of dose of an inhibitor having the
structure of
Formula I-XV on cognitive deficits and difference between amnestic Mild
Cognitive
Impairment patients treated with the inhibitor, and amnestic Mild Cognitive
Impairment
patients treated with donepezil. Improvement in Mini-Mental State Exam (MMSE),
Dementia Rating Scale (DRS) or Auditory Verbal Learning Test (AVLT) scores are
the
primary outcome measures of this study.
Secondary Outcome Measures:
[00587] To determine if the inhibitor having the structure of Formula I-XV has
comparable or better efficacy for treating cognitive deficits of amnestic Mild
Cognitive
Impairment compared to efficacy of donepezil for treating cognitive deficits
of amnestic
Mild Cognitive Impairment.
Inclusion Criteria:
[00588] Subjects between ages 55-80, both males and females. Diagnosis of
amnestic
Mild Cognitive Impairment. Had a CT scan or MRI scan within the prior 12
months, which
is compatible with a diagnosis of probable amnestic Mild Cognitive Impairment.
Asymptomatic with regard to dementia. MMSE scores of 21-24.
Exclusion Criteria:
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[00589] Significant neurological disease including Alzheimer's disease,
cerebral tumor,
Huntington's Disease, Parkinson's Disease, normal pressure hydrocephalus, or
other
diseases. Abnormal laboratory tests that might point to another etiology for
dementia: serum
B 12, folate, thyroid functions, electrolytes, syphilis serology.
Musculoskeletal diseases that
could interfere with assessment. Use of any drug within 14 days prior to
randomization
unless the dose of the drug and the condition being treated have been stable
for at least 30
days and are expected to remain stable during the study and neither the drug
nor the
condition being treated is expected to interfere with the study endpoints.
Experimental Design
[00590] Patients are divided into two groups, a donepezil group and a
PAK1/PAK3
inhibitor group. Each patient receives two daily doses of donepezil or a
PAK1/PAK3
inhibitor. Patients are monitored for a period of 40 weeks with experimental
sessions every
4 weeks.
[00591] Subjects are seated in a chair for each experimental session that
lasts about 3 h.
Surface electromygraphy (EMG) is recorded from the right abductor pollicis
brevis (APB)
muscle with disposable disc electrodes placed in a tendon-belly arrangement
over the bulk
of the APB muscle and the first metacarpal-phalangeal joint. The EMG is
monitored on a
computer screen, the signal is amplified and stored in a laboratory computer
for off-line
analysis. Transcranial magnetic stimulation (TMS) is performed with a Magstim
200
stimulator placed at an optimal position on the APB muscle. Electric
stimulation of the right
median nerve is performed with a stimulation block using constant current
square wave
pulses with cathode positioned proximally. The stimulus intensity delivered is
300% of the
sensory threshold.
[00592] Cortical excitability and cortical inhibition is measured prior to and
after Paired
Associative Stimulation (PAS). PAS consists of electric stimuli delivered to
the right
median nerve, paired with single pulse transcranial magnetic stimulation (TMS)
over
contralateral M1, with median nerve stimulation preceding TMS with
interstimulus interval
of 25 ms. Pairs of TMS and electrical stimuli are delivered at 0.1 hz over a
30 min period,
reaching a total of 180 pairs. Cortical excitability is measured using motor
evoked potentials
(MEPs) size which is defined as intensity of stimulus sufficient to produce a
mean MEP
amplitude of 1 mV peak-to-peak response at baseline (stimulus intensity of
Shmv). Cortical
inhibition is measured using cortical silent period (CSP). The CSP duration is
the time from
MEP onset to return of voluntary EMG activity.
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[00593] Patients are evaluated at weekly visits over a period of 40 weeks.
Groups are
compared using ANOVA. Single variable differences are analyzed using an
independent
samples t-test. A Pearson's coefficient is used to determine relationship
between cognition
and medication dose. Clinical Global Impressions (CGI) score, performance on
Mini-
Mental State Exam (MMSE), Dementia Rating Scale (DRS), Boston Naming Test,
Stroop
Color Word Test, Trail Making Test or Auditory Verbal Learning Test (AVLT) are
scored
at each visit. Clinician's Interview-Based Impression of Change are also
recorded at each
visit.
Example 62 Clinical Trial: Treatment of Autism with a PAK Inhibitor
l0 [00594] The following human clinical trial is performed to determine the
safety and
efficacy of a PAK inhibitor compound of Formula I-XV described herein for the
treatment
of autistic spectrum disorders. The study aims to provide preliminary
estimates of effect of
administration of a PAK inhibitor (of Formula I-XV described herein) in
alleviating,
inhibiting the progression of, or reducing the severity of at least one
behavioral symptom
associated autistic spectrum disorders over a three month study period.
Clinical
observations of global function in language and/or behavior pattern are
assessed.
[00595] Twenty-four patients, including 20 males and 4 females with an average
age of 9
years and meeting DSM-IV criteria for ASD, are treated with a compound of
Formula I-XV
described herein for up to three months. Patients assigned to the Experimental
group will
receive 1.5 mg twice a day for the first 2 weeks, 3 mg twice a day over the
next 2 weeks, 4.5
mg twice a day dose for the next 2 weeks and then 6 mg twice a day for the
remaining
period so at the time of the 12 weeks behavioral assessments, all patients are
on the
maximum dose.
[00596] The patients are evaluated using a global clinical improvement scale
rating for
improvement in language and behaviors based on parental observation and
clinical
appearance. Improvements are rated as follows: moderate to significant, mild
to moderate,
or no improvement.
[00597] After the twenty-four patients are treated for more up to three months
with a
compound of Formula I-XV described herein, parents report improvements in 20
of the 24
patients in one or more categories: attention, motor planning, language
function (both
receptively and expressively), and self-stimulatory behaviors.
Example 63 Clinical Trial to Evaluate the Safety of a Compound of Formula I-XV
in
Individuals with Neurofibromatosis Type I (NF1)
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[00598] Purpose: Neurofibromatosis type I (NFI) is a genetic disorder that
affects
approximately 1 in 3500 individuals. Half of people with NF1 inherit the
condition from a
parent, and half have a new occurrence of the condition. The manifestation of
NF 1 is highly
variable and multiple organ systems are typically affected. Some of the more
common
symptoms include benign neurofibromas, cafe an lait spots, Lisch nodules (tan
spots on the
iris of the eye). Some individuals with NF1 also exhibit more severe
associated conditions,
such as optic pathway tumors (gliomas) or bones bending or curving.
Neurocognitive
deficits and specific learning disabilities occur in approximately 30 to 50%
of individuals
with NF1 and are regarded by some observers and sufferers to be among the most
troubling
features of a disease. The most commonly reported findings are deficits in
visuoperceptual
ability, motor coordination, expressive and receptive language, and executive
functioning,
which requires intact short-term memory and attention. Patients with NF1 also
show a slight
depression in mean IQ scores compared to healthy adults without the disorder.
[00599] While cognitive deficits are now a widely-recognized feature of
neurofibromatosis type I (NF I), the precise cause of these deficits still
remain to be
determined.
[00600] A randomized, double-blinded, placebo- controlled, trial of a compound
of
Formula I-XV in patients with NF 1. Participants are randomly assigned to a
compound of
Formula I-XV or placebo and treated for approximately 14 weeks with baseline
and follow-
up assessments to evaluate safety and any effects on neurocognitive test
performance.
[00601] Study Type: Interventional
[00602] Design: Placebo Control; Endpoint Classification: Safety and Efficacy
study
[00603] Primary Outcome Measures: Non-verbal learning [Time Frame: 14 weeks]
[00604] Secondary Outcome Measures: attention [Time Frame: 14 weeks];
tolerability of
medication [Time Frame: 14 weeks]
[00605] Estimated Enrollment: 50
[00606] Eligibility: 10 years to 45 years; genders eligible for study: both
[00607] Inclusion Criteria:
a. a diagnosis of NF 1 by NIH criteria
b. between 10 and 45 years of age
c. no evidence of a comorbid neurological disorder (e.g., epilepsy,
encephalitis)
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d. not suffering from hypercholesterolemia based on self-report, collateral
information from physician, or initial medical workup using National
Cholesterol Education Program (NCEP, JAMA 2001), guidelines
accepted by the American College of Cardiology (ACC) and the
American Heart Association (AHA)
e. no mental retardation (i.e., IQ greater than 70)
f. no evidence of significant and habitual alcohol or drug abuse or
dependence
g. sufficient acculturation and fluency in the English language to avoid
invalidating research measures of thought, language, and speech disorder,
and verbal abilities
[00608] Exclusion Criteria:
a. comorbid neurological conditions
b. significant drug or alcohol abuse
c. non-fluency in English
Example 64: Clinical Trial to Evaluate the Safety of a Compound Described
Herein in
Patients with Imatinib-resistant Chronic Myelogenous Leukemia (CML)
[00609] Pu ose: The purpose of this trial is to assess the efficacy, safety,
tolerability,
biologic activity, and pharmacokinetics of a compound described herein in
patients with one
of the following conditions:
[00610] Imatinib failure only:imatinib-resistant or intolerant CML - Chronic
Phase (CP)
[00611] Imatinib-resistant or intolerant CML - Accelerated Phase (AP)
[00612] Imatinib-resistant or intolerant CML - Blast Crisis (BC)
[00613] Primary Outcome Measures:
[00614] To determine the maximum tolerated dose (MTD) and dose-limiting
toxicity
(DLT) of a compound described herein as a single agent when administered as an
oral once-
daily and twice daily dose to adult patients with imatinib-resistant CML
[00615] To characterize the pharmacokinetic profile of a compound described
herein in
serum and, where samples are available, in tumor cells and normal
hematopoietic cells
[00616] To evaluate the efficacy and safety of a compound described herein in
patients
with imatinib-resistant or intolerant CML-BC, imatinib-resistant or intolerant
CML-AP and
imatinib-resistant or intolerant CML-CP
[00617] Secondary Outcome Measures:
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[00618] To assess changes during and after therapy in malignant cells taken
from the
bone marrow and/or blood
[00619] To evaluate the population pharmacokinetics of a compound described
herein
[00620] To examine whether individual genetic variation in genes relating to
drug
metabolism, CML and the drug pathway confer differential response to a
compound
described herein
[00621] To identify gene expression patterns in tumor cells that are
associated with
treatment response to a compound escribed herein or that correlate with the
severity or
progression of CML
[00622] Eligibility: All genders 18 years and older
[00623] Criteria
a. Inclusion Criteria:
i. Main inclusion criteria include:
1. Patients with CML in blast crisis, CML in accelerated
phase defined as never in blast crisis phase, or CML in
chronic phase defined as never been in blast crisis phase
or accelerated phase who have: *developed progressive
disease during therapy with at least 600 mg of imatinib
per day, -OR- *patients with CML on imatinib therapy, at
any dose, developing progressive disease and the presence
of a genetic mutation likely to result in imatinib resistance
-OR- *have developed an intolerance to imatinib
2. CML patients who have been treated with an
investigational tyrosine kinase inhibitor who otherwise
meet the definition of imatinib-resistance or intolerance
are eligible
3. Written informed consent prior to any study procedures
being performed
b. Exclusion Criteria:
i. Impaired cardiac function
ii. Patients with severe/chronic or uncontrolled medical conditions
(including but not limited to diabetes, infections, GI impairment,
CNS infiltration, liver and kidney disease)
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iii. Prior and concomitant use of certain medications (including but
not limited to warfarin, chemotherapy, hematopoietic colony-
stimulating growth factors, medications that can affect
electrocardiogram test results, other investigational drugs)
iv. Women who are pregnant or breastfeeding
v. Patients with a history of another primary malignancy that is
currently clinically significant or currently requires active
intervention
vi. Patients unwilling to comply with the protocol
vii. Known diagnosis of human immunodeficiency virus (HIV)
infection
Example 65: Pharmaceutical Compositions
Example 65a: Parenteral Composition
[00624] To prepare a parenteral pharmaceutical composition suitable for
administration
by injection, 100 mg of a water-soluble salt of a compound of Formula I-XV is
dissolved in
DMSO and then mixed with 10 mL of 0.9% sterile saline. The mixture is
incorporated into
a dosage unit form suitable for administration by injection.
Example 65b: Oral Composition
[00625] To prepare a pharmaceutical composition for oral delivery, 100 mg of a
compound of Formula I-XV is mixed with 750 mg of starch. The mixture is
incorporated
into an oral dosage unit for, e.g., a hard gelatin capsule, which is suitable
for oral
administration.
Example 65c: Sublingual (Hard Lozenge) Composition
[00626] To prepare a pharmaceutical composition for buccal delivery, such as a
hard
lozenge, mix 100 mg of a compound of Formula I-XV with 420 mg of powdered
sugar
mixed, with 1.6 mL of light corn syrup, 2.4 mL distilled water, and 0.42 mL
mint extract.
The mixture is gently blended and poured into a mold to form a lozenge
suitable for buccal
administration.
Example 65d: Fast-Disintegrating Sublingual Tablet
[00627] A fast-disintegrating sublingual tablet is prepared by mixing 48.5% by
weigh of
a compound of Formula I-XV, 44.5% by weight of microcrystalline cellulose (KG-
802), 5%
by weight of low-substituted hydroxypropyl cellulose (50 m), and 2% by weight
of
magnesium stearate. Tablets are prepared by direct compression (AAPS
PharmSciTech.
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2006;7(2):E41). The total weight of the compressed tablets is maintained at
150 mg. The
formulation is prepared by mixing the amount of compound of Formula I-XV with
the total
quantity of microcrystalline cellulose (MCC) and two-thirds of the quantity of
low-
substituted hydroxypropyl cellulose (L-HPC) by using a three dimensional
manual mixer
(lnversina , Bioengineering AG, Switzerland) for 4.5 minutes. All of the
magnesium
stearate (MS) and the remaining one-third of the quantity of L-HPC are added
30 seconds
before the end of mixing.
Example 65e: Inhalation Composition
[00628] To prepare a pharmaceutical composition for inhalation delivery, 20 mg
of a
compound of Formula I-XV is mixed with 50 mg of anhydrous citric acid and 100
mL of
0.9% sodium chloride solution. The mixture is incorporated into an inhalation
delivery unit,
such as a nebulizer, which is suitable for inhalation administration.
Example 65f: Rectal Gel Composition
[00629] To prepare a pharmaceutical composition for rectal delivery, 100 mg of
a
compound of Formula I-XV is mixed with 2.5 g of methylcellulose (1500 mPa),
100 mg of
methylparapen, 5 g of glycerin and 100 mL of purified water. The resulting gel
mixture is
then incorporated into rectal delivery units, such as syringes, which are
suitable for rectal
administration.
Example 65g: Topical Gel Composition
[00630] To prepare a pharmaceutical topical gel composition, 100 mg of a
compound of
Formula I-XV is mixed with 1.75 g of hydroxypropyl cellulose, 10 mL of
propylene glycol,
10 mL of isopropyl myristate and 100 mL of purified alcohol USP. The resulting
gel
mixture is then incorporated into containers, such as tubes, which are
suitable for topical
administration.
Example 65h: Ophthalmic Solution Composition
[00631] To prepare a pharmaceutical ophthalmic solution composition, 100 mg of
a
compound of Formula I-XV is mixed with 0.9 g of NaCl in 100 mL of purified
water and
filtered using a 0.2 micron filter. The resulting isotonic solution is then
incorporated into
ophthalmic delivery units, such as eye drop containers, which are suitable for
ophthalmic
administration.
Example 65i: Nasal spray solution
[00632] To prepare a pharmaceutical nasal spray solution, 10 g of a compound
of
Formula I-XV is mixed with 30 mL of a 0.05M phosphate buffer solution (pH
4.4). The
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solution is placed in a nasal administrator designed to deliver 100 l of
spray for each
application.
[00633] While some embodiments of the present disclosure have been shown and
described herein, such embodiments are provided by way of example only. It is
intended
that the following claims define the scope of the present disclosure and that
methods and
structures within the scope of these claims and their equivalents be covered
thereby.
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